DNA encoding a human serotonin receptor (5-HT4B) and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid encoding a mammalian 5-HT 4B  receptor and an isolated nucleic acid encoding a human 5-HT 4B  receptor, an isolated protein which is a mammalian 5-HT 4B  receptor, vectors comprising an isolated nucleic acid encoding a mammalian 5-HT 4B  receptor, mammalian cells comprising such vectors, antibodies directed to the 5-HT 4B  receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian or human 5-HT 4B  receptor, antisense oligonucleotides complementary to any sequences of a nucleic acid which encodes a mammalian or human 5-HT 4B  receptor, and pharmaceutical compounds related to the human 5-HT 4B  receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatments for alleviating abnormalities associated with a human 5-HT 4B  receptor.

BACKGROUND OF THE INVENTION

[0001] Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0002] The subject invention of this application, DNA encoding a human serotonin receptor (5-HT_(4B)) and uses thereof, refers to the “5-HT_(4B) receptor” which has been renamed to the “5-HT₇ receptor” by the “Serotonin Receptor Nomenclature Committee” of the IUPHAR. Thus, all claims herein directly or indirectly related to the 5-HT_(4B) receptor and nucleic acid molecules encoding the 5-HT_(4B) receptor are drawn to the 5-HT₇ receptor and nucleic acid molecules encoding the 5-HT₇ receptor also.

[0003] Primary amino acid sequence and signal transduction data are now published for four cloned 5-HT₁-like receptors, three cloned 5-HT₂ receptors, and one 5-HT₃ receptor. Analysis of the sequence homology as well as the signal transduction pathways of these receptors leads to their grouping on the basis of these attributes: The 5-HT₁ subfamily includes: 5-HT_(1A) (Fargin, 1988; Kobilka, 1989), 5-HT_(1B)/5-HT_(1Dβ) (Weinshank et al., 1991 and the rest), 5-HT_(1Dα) (Branchek et al. 1991; Hamblin and Metcalf, 1991; Weinshank et al., 1992), 5-HT_(1E) (Levy et al., 1992; McAllister et al., 1992, Zgombick et al., 1992) and S-HT_(1F) (Adham et al, 1993). These subtypes share >50% transmembrane amino acid identity and couple to the inhibition of adenylate cyclase. The 5-HT₂ family includes the 5-HT₂ receptor (Pritchett et al., 1988), 5-HT_(1C) (Julius et al., 1989) and 5-HT_(2F) (Rat Stomach Fundus; Foquet et al., 1992; Kursar et al., 1992). These receptors share >70% amino acid identity and coupling to phosphoinositide hydrolysis. The 5-HT₃ receptor has been shown to be a ligand-gated ion channel (Maricq et al., 1991). Heterogeneity of 5-HT₃ receptors is controversial, although other ligand-gated ion channels display significant heterogeneity. Notably absent from this series are the 5-HT₄ receptors. The second messenger coupling from tissue studies indicates activation of adenylate cyclase as a primary mode of signal transduction (Dumius et al., 1988; Bockaert et al., 1990). We report here the cloning of the first mammalian 5-HT receptor that couples to the stimulation of adenylate cyclase activity which we propose to name 5-HT_(4B). The pharmacological properties of this receptor indicate that it may be similar to a series of functionally defined 5-HT receptors described in the porcine vena cava (Trevethick et al., 1984), cat saphenous vein, coronary arteries [Cushing and Cohen, 1992, and several vascular dilatory effects (Mylecharane and Phillips, 1989). These receptors appear to underlie contractile and relaxant responses in isolated blood vessels indicating potential therapeutic benefit in angina, coronary artery disease, atherosclerosis, and possibly cerebral blood vessel disorders leading to stroke. The presence of this subtype in the CNS also indicates potential use in disorders of higher cognitive processes as well as control of autonomic function.

SUMMARY OF THE INVENTION

[0004] This invention provides an isolated nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor. In a preferred embodiment of this invention, the isolated nucleic acid encodes a human 5-HT4B receptor. In another embodiment of this invention, the nucleic acid molecule comprises a plasmid designated pcEXV-5-HT_(4B) (ATCC Accession No. 75332).

[0005] This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor. This invention also provides a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT_(4B) receptor.

[0006] This invention provides an antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a mammalian 5-HT_(4B) receptor so as to prevent translation of the mRNA molecule. This invention also provides an antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a human 5-HT_(4B) receptor so as to prevent translation of the mRNA molecule.

[0007] This invention provides a monoclonal antibody directed to a mammalian 5-HT_(4B) receptor. This invention also provides a monoclonal antibody directed to a human 5-HT4B receptor.

[0008] This invention provides a pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a mammalian 5-HT_(4B) receptor and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of mammalian 5-HT^(4B) receptor and a pharmaceutically acceptable carrier.

[0009] This invention provides a pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human 5-HT_(4B) receptor and a pharmaceutically acceptable carrier. This invention also provides pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.

[0010] This invention provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian 5-HT_(4B) receptor so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA encoding the mammalian 5-HT_(4B) receptor and when hybridized to mRNA encoding the mammalian 5-HT_(4B) receptor, the complementary mRNA reduces the translation of the mRNA encoding the mammalian 5-HT_(4B) receptor.

[0011] This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human 5-HT_(4B) so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA encoding the human 5-HT_(4B) and when hybridized to mRNA encoding the human 5-HT4B, the complementary mRNA reduces the translation of the mRNA encoding the human 5-HT4B.

[0012] This invention provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian 5-HT_(4B) receptor positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the mammalian 5-HT_(4B) receptor and when hybridized to mRNA encoding the 5-HT_(4B) receptor, the antisense mRNA thereby prevents the translation of mRNA encoding the 5-HT_(4B) receptor.

[0013] This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human 5-HT4B receptor positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human 5-HT_(4B) receptor and when hybridized to mRNA encoding human 5-HT_(4B) receptor, the antisense mRNA thereby prevents the translation of mRNA encoding the 5-HT_(4B) receptor.

[0014] This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a mammalian 5-HT_(4B) receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a mammalian 5-HT_(4B) receptor, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a mammalian 5-HT_(4B) receptor.

[0015] This invention provides a method of screening drugs to identify drugs which specifically interact with, and bind to, a human 5-HT_(4B) receptor on the surface of a cell which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HT_(4B) receptor, the protein encoded thereby is expressed on the cell surface, with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, a human 5-HT_(4B) receptor.

[0016] This invention also provides a method of determining the physiological effects of expressing varying levels of a mammalian 5-HT_(4B) receptor which comprises producing a transgenic nonhuman animal whose levels of mammalian 5-HT_(4B) receptor expression are varied by use of an inducible promoter which regulates mammalian 5-HT_(4B) receptor expression.

[0017] This invention also provides a method of determining the physiological effects of expressing varying levels of a human 5-HT_(4B) receptor which comprises producing a transgenic nonhuman animal whose levels of human 5-HT_(4B) receptor expression are varied by use of an inducible promoter which regulates human 5-HT^(4B) receptor expression.

[0018] This invention further provides a method of determining the physiological effects of expressing varying levels of mammalian 5-HT_(4B) receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of mammalian 5-HT_(4B) receptor.

[0019] This invention further provides a method of determining the physiological effects of expressing varying levels of human 5-HT_(4B) receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT_(4B) receptor.

[0020] This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a human 5-HT_(4B) receptor allele which comprises: a.) obtaining DNA of subjects suffering from the disorder; b.) performing a restriction digest of the DNA with a panel of restriction enzymes; c.) electrophoretically separating the resulting DNA fragments on a sizing gel; d.) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a 5-HT_(4B) receptor and labelled with a detectable marker; e.) detecting labelled bands which have hybridized to the DNA encoding a 5-HT_(4B) receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f.) preparing DNA obtained for diagnosis by steps a-e; and g.) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.

[0021] This invention provides a method for determining whether a substrate not known to be capable of binding to a can bind to a mammalian 5-HT_(4B) receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the mammalian 5-HT_(4B) receptor with the substrate under conditions permitting binding of substrates known to bind to the 5-HT_(4B) receptor, detecting the presence of any of the substrate bound to the 5-HT_(4B) receptor, and thereby determining whether the substrate binds to the 5-HT_(4B) receptor.

[0022] This invention provides a method for determining whether a substrate not known to be capable of binding to a human 5-HT_(4B) receptor can bind to a human 5-HT_(4B) receptor, which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding the human 5-HT_(4B) receptor with the substrate under conditions permitting binding of substrates known to bind to a human 5-HT_(4B) receptor, detecting the presence of any of the substrate bound to the 5-HT_(4B) receptor, and thereby determining whether the substrate binds to the human 5-HT_(4B) receptor.

FIGURE LEGENDS

[0023]FIG. 1. Nucleotide Sequence and Deduced Amino Acid Sequence of a Novel Human 5-HT_(4B) Serotonin Receptor. Nucleotides are presented in the 5′ to 3′ orientation and the coding region is numbered starting from the initiating methionine and ending in the termination codon. Deduced amino acid sequence by translation of a long open reading frame is shown, along with the 5′ and 3′ untranslated regions. Numbers in the left and right margins represent nucleotide (top line) and amino acid (bottom line) numberings, starting with the first position as the adenosine (A) and the initiating methionine (M), respectively.

[0024]FIG. 2. Sequence Alignment of the Human 5-HT_(4B) receptor clone (designated hp78a) with 5HT_(1A), 5HT_(1Dα), 5HT_(1Dβ), 5HT_(1E), and 5HT_(1F). The deduced amino acid sequence of the human 5-HT_(4B) receptor (first line), from the starting methionine (M) to the stop codon (*), is aligned with the human 5HT_(1A) serotonin receptor clone (Kobilka et al., 1987), 5HT_(1Dα) serotonin receptor clone (Hamblin et al., 1991; Weinshank et al., 1992; Demchyshyn et al., 1992), 5HT_(1Dβ) serotonin receptor clone (Jin et al., 1992; Weinshank et al., 1992), 5HT_(1E) serotonin receptor clone (Zgombick et al., 1992; McAllister et al., 1992), and 5HT_(1F) serotonin receptor clone (Adham et al., submitted). Hyphens represent added spaces necessary for proper alignment. Gray shading indicates residues in receptor clones which are identical to the human 5-HT4B receptor. Numbers above amino acid sequences correspond to amino acid positions of the human 5-HT4B receptor, starting with the initiating methionine (M) and ending with the termination codon (*), and including spaces to account for proper alignment.

[0025]FIG. 3. Human tissue distribution of mRNA coding for 5-HT_(4B) receptor gene. Total RNA isolated from various human tissues were converted to single-stranded cDNA by random-priming with reverse transcriptase. cDNAs were amplified by PCR using functional hp78a-specific PCR primers. PCR products were run on a 1.5% agarose gel, blotted onto nylon membranes and hybridized to internal gene-specific oligonucleotides. Positive control consisted of gene-specific recombinant plasmid; dH₂O served as a negative control. PCR amplification and Southern blotting of RNA samples not treated with reverse transcriptase were negative.

[0026]FIG. 4. Stimulation of cAMP production by 5-HT in transiently transfected Cos-7 cells expressing the cloned human 5-HT_(4B) receptor. cAMP measurements on intact cells were as described under Methods and Materials. Each data point represents the mean of triplicates from a single experiment representative of at least 2 others. The vertical bars indicate S.E.M. Data are presented as percent basal cAMP released (basal, 0.053±0.004 pmol/ml/10 min).

[0027]FIG. 5. Stimulation of cAMP production by 5-CT in transiently transfected Cos-7 cells expressing the cloned human 5-HT_(4B) receptor. cAMP measurements on intact cells were as described under Methods and Materials. Each data point represents the mean of triplicates from a single experiment representative of at least 2 others. The vertical bars indicate S.E.M. Data are presented as percent basal cAMP released (basal, 0.053±0.004 pmol/ml/10 min).

DETAILED DESCRIPTION OF THE INVENTION

[0028] This invention provides an isolated nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor. This invention further provides an isolated nucleic acid molecule encoding a human 5-HT_(4B) receptor. As used herein, the term “isolated nucleic acid molecule” means a non-naturally occurring nucleic acid molecule that is, a molecule in a form which does not occur in nature. Examples of such an isolated nucleic acid molecule are an RNA, cDNA, or isolated genomic DNA molecule encoding a mammalian 5-HT_(4B) receptor or a human 5-HT_(4B) receptor. As used herein, “5-HT_(4B) receptor” means a molecule which, under physiologic conditions, is substantially specific for the neurotransmitter serotonin, is saturable, of high affinity for serotonin and the activation of which is coupled to the activation of adenylate cyclase. One embodiment of this invention is an isolated nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor. Another, preferred embodiment is an isolated nucleic acid molecule encoding a human 5-HT_(4B) receptor. Such a molecule may have coding sequences substantially the same as the coding sequences shown in FIG. 1. The DNA molecule of FIG. 1 (Seq. I.D. No. 1) encodes the sequence of a human 5-HT_(4B) receptor. One means of isolating a mammalian 5-HT_(4B) receptor is to probe a mammalian genomic library with a natural or artificially designed DNA probe, using methods well known in the art. In the preferred embodiment of this invention, the mammalian 5-HT_(4B) receptor is a human protein and the nucleic acid molecule encoding the human 5-HT_(4B) receptor is isolated from a human genomic library and human cDNA library. Overlapping transmembrane oligonucleotide probes derived from the Drosophila serotonin receptor gene DRO5HTR are useful probes for this purpose. DNA and cDNA molecules which encode the human 5-HT_(4B) receptor are used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries, by methods described in more detail below. Transcriptional regulatory elements from the 5′, untranslated region of the isolated clone, and other stability, processing, transcription, translation, and tissue specificity determining regions from the 3′ and 5′ untranslated regions of the isolated gene are thereby obtained.

[0029] This invention provides an isolated nucleic acid molecule which has been so mutated as to be incapable of encoding a molecule having normal 5-HT_(4B) receptor activity, and not expressing native 5-HT_(4B) receptor. An example of a mutated nucleic acid molecule provided by this invention is an isolated nucleic acid molecule which has an in-frame stop codon inserted into the coding sequence such that the transcribed RNA is not translated into protein.

[0030] This invention further provides a cDNA molecule encoding a mammalian 5-HT_(4B) receptor, wherein the cDNA molecule has a coding sequence substantially the same as the coding sequence shown in FIG. 1 (Sequence I.D. No. 1). This invention also provides a cDNA molecule encoding a human 5-HT_(4B) receptor, wherein the cDNA molecule has a coding sequence substantially the same as the coding sequence shown in FIG. 1. (Sequence I.D. No. 1). These molecules and their equivalents were obtained by the means described above.

[0031] This invention also provides an isolated protein which is a mammalian 5-HT_(4B) receptor. In a preferred embodiment of this invention, the protein is a human 5-HT_(4B) receptor protein having an amino acid sequence substantially similar to the amino acid sequence shown in FIG. 1 (Seq. I.D. Nos. 1 and 2). In another embodiment of this invention, the protein is a murine 5-HT_(4B) receptor protein having an amino acid sequence substantially similar to the amino acid sequence shown in FIG. 1 (Seq. I.D. Nos.1 and 2). As used herein, the term “isolated protein” is intended to encompass a protein molecule free of other cellular components. One means for obtaining an isolated mammalian 5-HT_(4B) receptor protein is to express DNA encoding the 5-HT_(4B) receptor in a suitable host, such as a bacterial, yeast, or mammalian cell, using methods well known to those skilled in the art, and recovering the receptor protein after it has been expressed in such a host, again using methods well known in the art. The receptor may also be isolated from cells which express it, in particular from cells which have been transfected with the expression vectors described below in more detail.

[0032] This invention provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA, encoding a mammalian 5-HT_(4B) receptor. This invention further provides a vector comprising an isolated nucleic acid molecule such as DNA, RNA, or cDNA, encoding a human 5-HT_(4B) receptor. Examples of vectors are viruses such as bacteriophages (including but not limited to phage lambda), animal viruses (including but not limited to Baculovirus, Murine leukemia virus, and Herpes virus), cosmids, plasmids (such as pUC18, available from Pharmacia, Piscataway, N.J.), and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known to those skilled in the art. Examples of such plasmids are plasmids comprising cDNA having a coding sequence substantially the same as the coding sequence shown in FIG. 1 (Seq. I.D. No. 1) and designated clone pcEXV-5HT_(4B) deposited under ATCC Accession No. 75332.

[0033] Alternatively, to obtain these vectors, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with a ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available.

[0034] This invention also provides vectors comprising a DNA molecule encoding a mammalian 5-HT_(4B) receptor and vectors comprising a DNA molecule encoding a human 5-HT_(4B) receptor, adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, mammalian, or insect cells so located relative to the DNA encoding a mammalian 5-HT_(4B) receptor or to the DNA encoding a human or mammalian 5-HT_(4B) receptor as to permit expression thereof. DNA having coding sequences substantially the same as the coding sequence shown in FIG. 1 may be usefully inserted into these vectors to express a human 5-HT_(4B) receptor. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982). Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Furthermore, an insect expression vector, such as recombinant Baculovirus, uses the polyhedrin gene expression signals for expression of the inserted gene in insect cells. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general. Expression vectors are useful to produce cells that express receptors. Certain uses for such cells are described in more detail below.

[0035] In one embodiment of this invention a plasmid is adapted for expression in a bacterial, yeast, insect or, in particular, a mammalian cell wherein the plasmid comprises a DNA molecule encoding a mammalian 5-HT_(4B) receptor or a DNA molecule encoding a human 5-HT_(4B) receptor and the regulatory elements necessary for expression of the DNA in the bacterial, yeast, insect or mammalian cell so located relative to the DNA encoding a mammalian 5-HT_(4B) receptor or to the DNA encoding a human 5-HT_(4B) receptor as to permit expression thereof. Suitable plasmids may include, but are not limited to plasmids adapted for expression in a mammalian cell, e.g., EVJB, EXV-3. An example of such a plasmid adapted for expression in a mammalian cell is a plasmid comprising cDNA having coding sequences substantially the same as the coding sequence shown in FIG. 1 (Seq I.D. No. 1) and the regulatory elements necessary for expression of the DNA in the mammalian cell. This plasmid has been designated pcEXV-5-HT_(4B) deposited under ATCC Accession No. 75332. Those skilled in the art will readily appreciate that numerous plasmids adapted for expression in a mammalian cell which comprise DNA encoding a mammalian or human 5-HT_(4B) receptor and the regulatory elements necessary to express such DNA in the mammalian cell may be constructed utilizing existing plasmids and adapted as appropriate to contain the regulatory elements necessary to express the DNA in the mammalian cell. The plasmids may be constructed by the methods described above for expression vectors and vectors in general, and by other methods well known in the art. Deposit discussed supra were made pursuant to, and in satisfaction of, the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852.

[0036] This invention provides a mammalian cell comprising a DNA molecule encoding a mammalian 5-HT_(4B) receptor, such as a mammalian cell comprising a plasmid adapted for expression in a mammalian cell, which comprises a DNA molecule encoding a mammalian 5-HT_(4B) receptor and the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a mammalian 5-HT_(4B) receptor as to permit expression thereof. This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT_(4B) receptor, such as a mammalian cell comprising a plasmid adapted for expression in a mammalian cell, which comprises a DNA molecule encoding a human 5-HT_(4B) receptor and the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a human 5-HT_(4B) receptor as to permit expression thereof. Numerous mammalian cells may be used as hosts, including, but not limited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, LM (tk−) cells, Cos cells, etc. Expression plasmids such as that described supra may be used to transfect mammalian cells by methods well known in the art such as calcium phosphate precipitation, or DNA encoding a human or mammalian 5-HT_(4B) receptor may be otherwise introduced into mammalian cells, e.g., by microinjection, to obtain mammalian cells which comprise DNA, e.g., cDNA or a plasmid, encoding a human or mammalian 5-HT_(4B) receptor. LM (tk−) cells comprising and expressing the plasmid pcEXV-5HT_(4B) were deposited with the ATCC as described hereinabove and given the ATCC Accession No. CRL 11166.

[0037] This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT_(4B) receptor, for example with a coding sequence included within the sequences shown in FIG. 1 (SEQ. I.D. NO.1) As used herein, the phrase “specifically hybridizing” means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. Detection of nucleic acid encoding a human 5-HT_(4B) receptor is useful as a diagnostic test for any disease process in which levels of expression of the 5-HT_(4B) receptor are altered. DNA probe molecules are produced by insertion of a DNA molecule which encodes a 5-HT_(4B) receptor or fragments thereof into suitable vectors, such as plasmids or bacteriophages, followed by insertion into suitable bacterial host cells and replication and harvesting of the DNA probes, all using methods well known in the art. For example, the DNA may be extracted from a cell lysate using phenol and ethanol, digested with restriction enzymes corresponding to the insertion sites of the DNA into the vector (discussed above), electrophoresed, and cut out of the resulting gel. An example of such DNA molecule is shown in FIG. 1. The probes are useful for ‘in situ’ hybridization or in order to locate tissues which express this gene family, or for other hybridization assays for the presence of these genes or their mRNA in various biological tissues. In addition, synthesized oligonucleotides (produced by a DNA synthesizer) complementary to the sequence of a DNA molecule which encode a mammalian 5-HT_(4B) receptor or complementary to the sequence of a DNA molecule which encodes a human 5-HT_(4B) receptor are useful as probes for these genes, for their associated mRNA, or for the isolation of related genes by homology screening of genomic or cDNA libraries, or by the use of amplification techniques such as the Polymerase Chain Reaction.

[0038] This invention also provides a method of detecting expression of a human 5-HT_(4B) receptor on the surface of a cell by detecting the presence of mRNA coding for a 5-HT_(4B) receptor. This invention further provides a method of detecting expression of a mammalian 5-HT_(4B) receptor on the surface of the cell by detecting the presence of mRNA coding for a mammalian 5-HT_(4B) receptor. These methods comprise obtaining total mRNA from the cell using methods well known in the art and contacting the mRNA so obtained with a nucleic acid probe as described, hereinabove, under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell. Hybridization of probes to target nucleic acid molecules such as mRNA molecules employs techniques well known in the art. However, in one embodiment of this invention, nucleic acids are extracted by precipitation from lysed cells and the mRNA is isolated from the extract using a column which binds the poly-A tails of the mRNA molecules (Maniatis, T. et al., Molecular Cloning; Cold Spring Harbor Laboratory, pp.197-98 (1982)). The mRNA is then exposed to radioactively labelled probe on a nitrocellulose membrane, and the probe hybridizes to and thereby labels complementary mRNA sequences. Binding may be detected by autoradiography or scintillation counting. However, other methods for performing these steps are well known to those skilled in the art, and the discussion above is merely an example.

[0039] This invention provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human 5-HT_(4B) receptor so as to prevent translation of the human 5-HT_(4B) receptor. This invention also provides an antisense oligonucleotide having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a mammalian 5-HT_(4B) receptor so as to prevent translation of the mammalian 5-HT_(4B) receptor. As used herein, the phrase “binding specifically” means the ability of an antisense oligonucleotide to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs. The antisense oligonucleotide may have a sequence capable of binding specifically with any sequences of the cDNA molecule whose sequence is shown in FIG. 1. A particular example of an antisense oligonucleotide is an antisense oligonucleotide comprising chemical analogues of nucleotides.

[0040] This invention also provides a pharmaceutical composition comprising an effective amount of the oligonucleotide described above effective to reduce expression of a human 5-HT_(4B) receptor by passing through a cell membrane and binding specifically with mRNA encoding the 5-HT_(4B) receptor in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. This invention further provides a pharmaceutical composition comprising an effective amount of the oligonucleotide described above effective to reduce expression of a mammalian 5-HT_(4B) receptor by passing through a cell membrane and binding specifically with mRNA encoding a mammalian 5-HT_(4B) receptor in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The oligonucleotide may be coupled to a substance which inactivates mRNA, such as a ribozyme. The pharmaceutically acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a transporter specific for a selected cell type and is thereby taken up by cells of the selected cell type. The structure may be part of a protein known to bind a cell-type specific transporter, for example an insulin molecule, which would target pancreatic cells. DNA molecules having a coding sequence substantially the same as the coding sequences shown in FIG. 1 may be used as the oligonucleotides of the pharmaceutical composition.

[0041] This invention provides a method of treating abnormalities which are alleviated by reduction of expression of 5-HT_(4B) receptor. This method comprises administering to a subject an effective amount of the pharmaceutical composition described above effective to reduce expression of the 5-HT_(4B) receptor by the subject.

[0042] This invention further provides a method of treating an abnormal condition related to 5-HT_(4B) receptor activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to reduce expression of the 5-HT_(4B) receptor by the subject. Examples of such abnormal conditions are angina, coronary artery disease, atherosclerosis, cerebral blood vessel disorders leading to stroke, irritable bowel syndrome or other disorders of grastrointestinal motility, CNS disorders, pain perception, affective disorders, disorders of higher cognitive processes including but not limited to schizophrenia, as well as control of autonomic function.

[0043] Antisense oligonucleotide drugs inhibit translation of mRNA encoding 5-HT_(4B) receptor. Synthetic antisense oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding the 5-HT_(4B) receptor and inhibit translation of mRNA and are useful as drugs to inhibit expression of 5-HT_(4B) receptor genes in patients. This invention provides a means to therapeutically alter levels of expression of a human or mammalian 5-HT_(4B) receptor by the use of a synthetic antisense oligonucleotide drug (SAOD) which inhibits translation of mRNA encoding the 5-HT_(4B) receptor. Synthetic antisense oligonucleotides, or other antisense chemical structures designed to recognize and selectively bind to mRNA, are constructed to be complementary to portions of the nucleotide sequence shown in FIG. 1 of DNA, RNA or of chemically modified, artificial nucleic acids. The SAOD is designed to be stable in the blood stream for administration to patients by injection, or in laboratory cell culture conditions, for administration to cells removed from the patient. The SAOD is designed to be capable of passing through cell membranes in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SAOD which render it capable of passing through cell membranes (e.g., by designing small, hydrophobic SAOD chemical structures) or by virtue of specific transport systems in the cell which recognize and transport the SAOD into the cell. In addition, the SAOD can be designed for administration only to certain selected cell populations by targeting the SAOD to be recognized by specific cellular uptake mechanisms which bind and take up the SAOD only within certain selected cell populations. For example, the SAOD may be designed to bind to transporter found only in a certain cell type, as discussed above. The SAOD is also designed to recognize and selectively bind to the target mRNA sequence, which may correspond to a sequence contained within the sequence shown in FIG. 1 by virtue of complementary base pairing to the mRNA. Finally, the SAOD is designed to inactivate the target mRNA sequence by any of three mechanisms: 1) by binding to the target mRNA and thus inducing degradation of the mRNA by intrinsic cellular mechanisms such as RNAse I digestion, 2) by inhibiting translation of the mRNA target by interfering with the binding of translation-regulating factors or of ribosomes, or 3) by inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups, which either degrade or chemically modify the target mRNA. Synthetic antisense oligonucleotide drugs have been shown to be capable of the properties described above when directed against mRNA targets (J. S. Cohen, Trends in Pharm. Sci. 10, 435 (1989); H. M. Weintraub, Sci. Am. January (1990) p. 40). In addition, coupling of ribozymes to antisense oligonucleotides is a promising strategy for inactivating target mRNA (N. Sarver et al., Science 247, 1222 (1990)). An SAOD serves as an effective therapeutic agent if it is designed to be administered to a patient by injection, or if the patient's target cells are removed, treated with the SAOD in the laboratory, and replaced in the patient. In this manner, an SAOD serves as a therapy to reduce 5-HT_(4B) receptor expression in particular target cells of a patient, in any clinical condition which may benefit from reduced expression of 5-HT_(4B) receptor.

[0044] This invention provides an antibody directed to the human 5-HT_(4B) receptor. This invention also provides an antibody directed to the mammalian 5-HT_(4B) receptor. This antibody may comprise, for example, a monoclonal antibody directed to an epitope of a human 5-HT_(4B) receptor present on the surface of a cell, the epitope having an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the human 5-HT_(4B) receptor included in the amino acid sequence shown in FIG. 1. Amino acid sequences may be analyzed by methods well known to those skilled in the art to determine whether they produce hydrophobic or hydrophilic regions in the proteins which they build. In the case of cell membrane proteins, hydrophobic regions are well known to form the part of the protein that is inserted into the lipid bilayer which forms the cell membrane, while hydrophilic regions are located on the cell surface, in an aqueous environment. Therefore antibodies to the hydrophilic amino acid sequences shown in FIG. 1 will bind to a surface epitope of a 5-HT_(4B) receptor as described. Antibodies directed to a human or mammalian 5-HT_(4B) receptor may be serum-derived or monoclonal and are prepared using methods well known in the art. For example, monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody. Cells such as NIH3T3 cells or LM (tk⁻) cells may be used as immunogens to raise such an antibody. Alternatively, synthetic peptides may be prepared using commercially available machines and the amino ac i sequences shown in FIG. 1. As a still further alternative, DNA, such as a cDNA or a fragment thereof, may be cloned and expressed and the resulting polypeptide recovered and used as an immunogen. These antibodies are useful to detect the presence of 5-HT_(4B) receptor encoded by the isolated DNA, or to inhibit the function of the 5-HT_(4B) receptor in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.

[0045] This invention also provides a pharmaceutical composition which comprises an effective amount of an antibody directed to an epitope of the human or mammalian 5-HT_(4B) receptor, effective to block binding of naturally occurring substrates to the 5-HT_(4B) receptor, and a pharmaceutically acceptable carrier. A monoclonal antibody directed to an epitope of a human 5-HT_(4B) receptor present on the surface of a cell which has an amino acid sequence substantially the same as an amino acid sequence for a cell surface epitope of the human 5-HT_(4B) receptor included in the amino acid sequence shown in FIG. 1 is useful for this purpose.

[0046] This invention also provides a method of treating abnormalities in a subject which are alleviated by reduction of expression of a human or mammalian 5-HT_(4B) receptor which comprises administering to the subject an effective amount of the pharmaceutical composition described above effective to block binding of naturally occurring substrates to the receptor and thereby alleviate abnormalities resulting from overexpression of a human or mammalian 5-HT_(4B)receptor. Binding of the antibody to the receptor prevents the receptor from functioning, thereby neutralizing the effects of overexpression. The monoclonal antibodies described above are useful for this purpose. This invention additionally provides a method of treating an abnormal condition related to an excess of 5-HT_(4B) receptor activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to block binding of naturally occurring substrates to the 5-HT_(4B) receptor and thereby alleviate the abnormal condition. Some examples of abnormal conditions associated with excess 5-HT_(4B) receptor activity are angina, coronary artery disease, atherosclerosis, cerebral blood vessel disorders leading to stroke, disorders of higher cognitive processes (e.g. schizophrenia) as well as control of autonomic function.

[0047] This invention provides methods of detecting the presence of a 5-HT_(4B) receptor on the surface of a cell which comprises contacting the cell with an antibody directed to the 5-HT_(4B) receptor, under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby the presence of the 5-HT_(4B) receptor on the surface of the cell. Such methods are useful for determining whether a given cell is defective in expression of 5-HT_(4B) receptors. Bound antibodies are detected by methods well known in the art, for example by binding fluorescent markers to the antibodies and examining the cell sample under a fluorescence microscope to detect fluorescence on a cell indicative of antibody binding. The monoclonal antibodies described above are useful for this purpose.

[0048] This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HT_(4B) receptor and a transgenic nonhuman mammal expressing DNA encoding a mammalian 5-HT_(4B) receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human or mammalian 5-HT_(4B) receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT_(4B) receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises DNA encoding a human 5-HT_(4B) receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a human 5-HT_(4B) receptor which hybridizes to mRNA encoding a 5-HT_(4B) receptor thereby reducing its translation and a transgenic nonhuman mammal whose genome comprises DNA encoding a mammalian 5-HT_(4B) receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a mammalian 50-HT_(4B) receptor and which hybridizes to mRNA encoding a mammalian 5-HT_(4B) receptor thereby reducing its translation. The DNA may additionally comprise an inducible promoter or additionally comprise tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of DNA are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequences shown in FIG. 1. An example of a transgenic animal is a transgenic mouse. Examples of tissue specificity-determining regions are the metallothionein promotor (Low, M. J., Lechan, R. M., Hammer, R. E. et al. Science 231:1002-1004 (1986)) and the L7 promotor (Oberdick, J., Smeyne, R. J., Mann, J. R., Jackson, S. and Morgan, J. I. Science 248:223-226 (1990)).

[0049] Animal model systems which elucidate the physiological and behavioral roles of mammalian receptors are produced by creating transgenic animals in which the expression of a receptor is either increased or decreased, or the amino acid sequence of the expressed receptor protein is altered, by a variety of techniques. Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a human 5-HT_(4B) receptor or homologous animal versions of this gene, by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)) or, 2) Homologous recombination (Capecchi M. R. Science 244:1288-1292 (1989); Zimmer, A. and Gruss, P. Nature 338:150-153 (1989)) of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of the receptor. The technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native receptor but does express, for example, an inserted mutant receptor, which has replaced the native receptor in the animal's genome by recombination, resulting in underexpression of the receptor. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added receptors, resulting in overexpression of the receptor.

[0050] One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium (Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding a receptor is puritied from a vector (such as plasmid pcEXV-5HT_(4B) described above) by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.

[0051] Since the normal action of receptor-specific drugs is to activate or to inhibit the receptor, the transgenic animal model systems described above are useful for testing the biological activity of drugs directed against the receptors even before such drugs become available. These animal model systems are useful for predicting or evaluating possible therapeutic applications of drugs which activate or inhibit receptors by inducing or inhibiting expression of the native or trans-gene and thus increasing or decreasing expression of normal or mutant receptors in the living animal. Thus, a model system is produced in which the biological activity of drugs directed against the receptors are evaluated before such drugs become available. The transgenic animals which over or under produce the receptor indicate by their physiological state whether over or under production of the receptor is therapeutically useful.

[0052] It is therefore useful to evaluate drug action based on the transgenic model system. One use is based on the fact that it is well known in the art that a drug such as an antidepressant acts by blocking neurotransmitter uptake, and thereby increases the amount of neurotransmitter in the synaptic cleft. The physiological result of this action is to stimulate the production of less receptor by the affected cells, leading eventually to underexpression. Therefore, an animal which underexpresses receptor is useful as a test system to investigate whether the actions of such drugs which result in under expression are in fact therapeutic. Another use is that if overexpression is found to lead to abnormalities, then a drug which down-regulates or acts as an antagonist to the receptor is indicated as worth developing, and if a promising therapeutic application is uncovered by these animal model systems, activation or inhibition of the 5-HT_(4B) receptor is achieved therapeutically either by producing agonist or antagonist drugs directed against the 5-HT_(4B) receptor or by any method which increases or decreases the expression of this receptor in man.

[0053] Further provided by this invention is a method of determining the physiological effects of expressing varying levels of human or mammalian 5-HT_(4B) receptors which comprises producing a transgenic nonhuman animal whose levels of human or mammalian 5-HT_(4B) receptor expression are varied by use of an inducible promoter which regulates receptor expression. This invention also provides a method of determining the physiological effects of expressing varying levels of human or mammalian 5-HT_(4B) receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human or mammalian 5-HT_(4B) receptor. Such animals may be produced by introducing different amounts of DNA encoding a human or mammalian 5-HT_(4B) receptor into the oocytes from which the transgenic animals are developed.

[0054] This invention also provides a method for identifying a substance capable of alleviating abnormalities resulting from overexpression of a human or mammalian 5-HT_(4B) receptor comprising administering the substance to a transgenic nonhuman mammal expressing at least one artificially introduced DNA molecule encoding a human or mammalian 5-HT_(4B) receptor and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of a human or mammalian 5-HT_(4B) receptor. As used herein, the term “substance” means a compound or composition which may be natural, synthetic, or a product derived from screening. Examples of DNA molecules are DNA or cDNA molecules having a coding sequence substantially the same as the coding sequences shown in FIG. 1.

[0055] This invention provides a pharmaceutical composition comprising an amount of the substance described supra effective to alleviate the abnormalities resulting from overexpression of 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.

[0056] This invention further provides a method for treating the abnormalities resulting from overexpression of a human or mammalian 5-HT_(4B) receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from overexpression of a human or mammalian 5-HT_(4B) receptor.

[0057] This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human or mammalian 5-HT_(4B) receptor comprising administering the substance to the transgenic nonhuman mammal described above which expresses only nonfunctional human or mammalian 5-HT_(4B) receptor and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of a human or mammalian 5-HT_(4B) receptor.

[0058] This invention also provides a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human or mammalian 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.

[0059] This invention further provides a method for treating the abnormalities resulting from underexpression of a human or mammalian 5-HT_(4B) receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to alleviate the abnormalities resulting from underexpression of a human or mammalian 5-HT_(4B) receptor.

[0060] This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a human or mammalian 5-HT_(4B) receptor allele which comprises: a) obtaining DNA of subjects suffering from the disorder; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c) electrophoretically separating the resulting DNA fragments on a sizing gel; d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human or mammalian 5-HT_(4B) receptor and labelled with a detectable marker; e) detecting labelled bands which have hybridized to the DNA encoding a the human or mammalian 5-HT_(4B) receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f) preparing DNA obtained for diagnosis by steps a-e; and g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and thereby to diagnose predisposition to the disorder if the patterns are the same. This method may also be used to diagnose a disorder associated with the expression of a specific 5-HT_(4B) human or mammalian 5-HT_(4B) receptor allele.

[0061] This invention provides a method of preparing the isolated 5-HT_(4B) receptor which comprises inducing cells to express receptor, recovering the receptor from the resulting cells, and purifying the receptor so recovered. An example of an 5-HT_(4B) receptor is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1. For example, cells can be induced to express receptors by exposure to substances such as hormones. The cells can then be homogenized and the receptor isolated from the homogenate using an affinity column comprising, for example serotonin or another substance which is known to bind to the 5-HT_(4B) receptor. The resulting fractions can then be purified by contacting them with an ion exchange column, and determining which fraction contains 5-HT_(4B) receptor activity or binds anti-receptor antibodies.

[0062] This invention provides a method of preparing the isolated 5-HT_(4B) receptor which comprises inserting nucleic acid encoding 5-HT_(4B) receptor in a suitable vector, inserting the resulting vector in a suitable host cell, recovering the receptor produced by the resulting cell, and purifying the receptor so recovered. An example of an isolated 5-HT_(4B) receptor is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1. These methods for preparing 5-HT_(4B) receptor uses recombinant DNA technology methods well known in the art. For example, isolated nucleic acid encoding 5-HT_(4B) receptor is inserted in a suitable vector, such as an expression vector. A suitable host cell, such as a bacterial cell, or a eukaryotic cell such as a yeast cell, is transfected with the vector. 5-HT_(4B) receptor is isolated from the culture medium by affinity purification or by chromatography or by other methods well known in the art.

[0063] This invention provides a method for determining whether a ligand not known to be capable of binding to a human 5-HT_(4B)receptor can bind to a human 5-HT4B receptor which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT_(4B) receptor, the protein encoded thereby is expressed on the cell surface, with the ligand under conditions permitting binding of ligands known to bind to the 5-HT receptors, detecting the presence of any of the ligand bound to the 5-HT_(4B) receptor, and thereby determining whether the ligand binds to the 5-HT_(4B) receptor. This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT_(4B) receptor can functionally activate its activity or prevent the action of a ligand which does so. This comprises contacting a mammalian cell comprising an isolated DNA molecule which encodes a human 5-HT_(4B) receptor with the ligand under conditions permitting the activation or blockade of a functional response, detected by means of a bioassay from the mammalian cell such as a second messenger response, and thereby determining whether the ligand activates or prevents the activation of the human 5-HT_(4B) receptor functional output. The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1 preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is an Ltk− cell, in particular the Ltk− cell designated L-5-HT_(4B). Another example of a non-neuronal mammalian cell to be used for functional assays is a murine fibroblast cell line, specifically the NIH3T3 cell. The preferred method for determining whether a ligand is capable of binding to the human 5-HT_(4B) receptor comprises contacting a transfected nonneuronal mammalian cell (i.e. a cell that does not naturally express any type of 5-HT or G-protein coupled receptor, thus will only express such a receptor if it is transfected into the cell) expressing a 5-HT_(4B) receptor on its surface, or contacting a membrane preparation derived from such a transfected cell, with the ligand under conditions which are known to prevail, and thus to be associated with, in vivo binding of the ligands to a 5-HT_(4B) receptor, detecting the presence of any of the ligand being tested bound to the 5-HT_(4B) receptor on the surface of the cell, and thereby determining whether the ligand binds to, activates or prevents the activation of the 5-HT_(4B) receptor. This response system is obtained by transfection of isolated DNA into a suitable host cell containing the desired second messenger system such as phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase or ion channels. Such a host system is isolated from pre-existing cell lines, or can be generated by inserting appropriate components of second messenger systems into existing cell lines. Such a transfection system provides a complete response system for investigation or assay of the activity of human 5-HT_(4B) receptor with ligands as described above. Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for these competitive binding assays. Functional assays of second messenger systems or their sequelae in transfection systems act as assays for binding affinity and efficacy in the activation of receptor function. A transfection system constitutes a “drug discovery system” useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the human 5-HT_(4B) receptor. The transfection system is also useful for determining the affinity and efficacy of known drugs at human 5-HT_(4B) receptor sites.

[0064] This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human or mammalian 5-HT_(4B) receptor on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human or mammalian 5-HT_(4B) receptor on the surface of a cell with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, the human and/or mammalian 5-HT_(4B) receptor. Various methods of detection may be employed. The drugs may be “labeled” by association with a detectable marker substance (e.g., radiolabel or a non-isotopic label such as biotin). The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in FIG. 1. Preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is a Cos7 cell. Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed 5-HT_(4B) receptor protein in transfected cells, using radioligand binding methods well known in the art, examples of which are shown in the binding assays described herein. Drug candidates are also screened for selectivity by identifying compounds which bind with high affinity to one particular receptor but do not bind with high affinity to any other receptor subtypes or to any other known receptor. Because selective, high affinity compounds interact primarily with the target 5-HT_(4B) receptor site after administration to the patient, the chances of producing a drug with unwanted side effects are minimized by this approach. This invention provides a pharmaceutical composition comprising a drug identified by the method described above and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. Once the candidate drug has been shown to be adequately bio-available following a particular route of administration, for example orally or by injection (adequate therapeutic concentrations must be maintained at the site of action for an adequate period to gain the desired therapeutic benefit), and has been shown to be non-toxic and therapeutically effective in appropriate disease models, the drug may be administered to patients by that route of administration determined to make the drug bio-available, in an appropriate solid or solution formulation, to gain the desired therapeutic benefit.

[0065] Applicants have identified a novel human 5HT receptor subtype protein, designated 5-HT_(4B) and have described methods for the identification of pharmacological compounds for therapeutic treatments. Pharmacological compounds which are directed against specific receptor subtypes provide effective new therapies with minimal side effects.

[0066] Elucidation of the molecular structures of the neuronal serotonin receptors is an important step in the understanding of serotonergic neurotransmission. This disclosure reports the isolation, amino acid sequence, and functional expression of a novel cDNA clone which encodes a human 5-HT_(4B) receptor. The identification of 5-HT receptor subtypes play a pivotal role in elucidating the molecular mechanisms underlying serotonergic transmission, and should also aid in the development of novel therapeutic agents.

[0067] A complementary DNA clone (designated hp78a) encoding a serotonin receptor subtype has been isolated from human a human cDNA and genomic DNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence of predicts a protein of 445 amino acids with 7 highly hydrophobic regions compatible with membrane-spanning domains. Analysis of 5-HT_(4B) structure and function provides a model for the development of drugs useful for the treatment of angina, coronary artery disease, atherosclerosis, cerebral blood vessel disorders leading to stroke, irritable bowel syndrome or other disorders of gastrointestinal motility, CNS disorders, pain perception, affective disorders, and disorders of higher cognitive processes including but not limited to schizophrenia, as well as control of autonomic function.

[0068] This invention identifies for the first time a mammalian serotonin receptor, its amino acid sequence, and its mammalian gene, the activation of which is information and experimental tools provided by this discovery are useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for this receptor protein, its associated mRNA molecule or its associated genomic DNA. The information and experimental tools provided by this discovery will be useful to generate new therapeutic agents, and new therapeutic or diagnostic assays for this new serotonin receptor subtype, its associated mRNA molecule, or its associated genomic DNA.

[0069] Specifically, this invention relates to the isolation of mammalian cDNA and genomic DNA clones encoding a new serotonin receptor, designated 5-HT_(4B). The new human gene for this receptor identified herein as hp78a has been identified and characterized and a series of related cDNA and genomic DNA clones have been isolated. In addition, the human 5-HT_(4B) receptor has been expressed in Cos7 cells by transfecting the cells with the plasmid pcEXV-5HT_(4B). The pharmacological binding properties of the encoded 5-HT_(4B) receptor have been determined, and the binding properties classify this receptor as a novel serotonin receptor. Mammalian cell lines expressing the human 5-HT_(4B) receptor on the cell surface has been constructed, thus establishing the first well-defined, cultured cell lines with which to study the novel 5-HT_(4B) receptor.

[0070] The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.

METHODS AND MATERIALS

[0071] Cloning and Sequencing: A human placenta genomic library in λ dash II (≈1.5×10⁶ total recombinants; Stratagene, LaJolla, Calif.) was screened using overlapping transmembrane (TM) oligonucleotide probes (TM 3, 5, 6 and 7) derived from the Drosophila serotonin receptor gene, Dro5HTR (Witz et al., 1990). Overlapping oligomers were labeled with [³²P]dATP and [³²P]dCTP by synthesis with the large fragment of DNA polymerase. Hybridization was performed at medium stringency conditions: 45° C. in a solution containing 37.5% formamide, 10% dextran sulfate, 5× SSC (1×SSC is 0.15M sodium chloride, 0.015M sodium citrate), 1× Denhardt's solution (0.02% polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin), and 200 μg/μl sonicated salmon sperm DNA. The filters were washed at 45° C. in 0.1× SSC containing 0.1% sodium dodecyl sulfate and exposed at −70° C. to Kodak XAR film in the presence of an intensifying screen. Lambda phage clones hybridizing with the probe were plaque purified and DNA was prepared for Southern blot analysis (Southern, 1975; Sambrook et al., 1989). For subcloning and further Southern blot analysis, DNA was cloned into pUC18 (Pharmacia, Piscataway, N.J.), pGEM-5Zf(Promega, Madison, Wis.) or pBluescriptII (Stratagene, LaJolla, Calif.). Nucleotide sequence analysis was accomplished by the Sanger dideoxy nucleotide chain termination method (Sanger et al., 1977) on denatured double-stranded plasmid templates, using Sequenase (US Biochemical Corp., Cleveland, Ohio), Bst DNA sequencing kit (Bio-Rad Laboratories, Richmond, Calif.), or TaqTrack sequencing kit (Promega Corporation, Madison, Wis.).

[0072] In order to isolate a full-length clone, human cDNA libraries were screened by polymerase chain reaction (PCR) with 1 μM each of specific oligonucleotide primers designed from the isolated genomic clone: from the sense strand (nucleotide 512-535), 5′TGACCCTGTGCGTGATCAGCATTG 3′ and from the anti-sense strand (nucleotide 945-974), 5′ GCTTTCTGTTCTCGCTTAAAGATGGAGATG 3′ (see FIG. 1). The primers were from the 3′ end of Tm3 and the Tm5/Tm6 loop regions for the upstream and downstream primers, respectively. One to 2 μl of phage DNA from cDNA libraries (λ ZapII; Stratagene, LaJolla, Calif.), representing ≈10⁶-10⁷ pfu, were amplified in 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM each dATP, dCTP, dGTP, dTTP, and 2.5 units of Thermus aquaticus DNA polymerase (Taq polymerase; Perkin-Elmer-Cetus, Norwalk, Conn.). The amplification profile was run for 30 cycles: a 5 min. initial (ie. 1 cycle) denaturation at 95° C., followed by 2 min. at 94° C., 2 min. at 68° C., and 3 min. at 72° C., with a 3 sec. extension, followed by a final 10 min. extension at 72° C. PCR products were analyzed by ethidium bromide (EtBr) stained agarose gels and any sample exhibiting a band on the EtBr stained gel was considered positive.

[0073] A positive library was then plated and screened with overlapping 45-mer oligonucleotide probes, filled-in using [α-³²P]dCTP and [α-³²P]dATP and Klenow fragment of DNA polymerase. This probe was internal to the amplification primers discussed above: from the sense strand (nucleotide 864-908), 5′ CCTGAATGGCATAGTGAAGCTCCAGAAGGAGGTGGAAGAGTGTGC 3′, and from the antisense strand (nucleotide 889-933), 5′ ATGCTTGAGGAGTCTCGAAAGGTTTGCACACTCTTCCACCTCCTT 3′ (see FIG. 1). Positive cDNA phage clones were plaque purified and pBluescript recombinant DNAs were excision-rescued from λ Zap II using helper phage R408, as described by manufacturer's protocol (Stratagene, Lajolla, Calif.). Insert size was confirmed by restriction enzyme digest analysis and recombinants were sequenced, as described above.

[0074] Three additional sets of overlapping oligonucleotides were used to isolate a total of three partial but overlapping cDNA clones. These oligonucleotides and corresponding cDNA clone names are: from hFB9a (in the Tm3/4 loop), the sense strand (nt. 529-573), 5′ AGCATTGACAGGTACCTTGGGATCACAAGGCCCCTCACATACCCT 3′, and the antisense strand (nt. 554-599), 5′ CCATGCATTTCCCATTCTGCCTCACAGGGTATGTGAGGGGCCTTG 3′; from hFB44a (in Tm 2/3 loop), the sense strand (nt. 412-456), 5′ GTCAGCGTCACCGACCTCATCGGGGGCAAGTGGATCTTTGGACAC 3′, and the antisense strand (nt. 437-481), 5′ TGGCGATGAAGACATTACAGAAAAAGTGTCCAAAGATCCACTTGC 3′; from hFB41a (in the NH₂ terminus), the sense strand (nt. 107-150), 5′ GGCGCCGACCCGGTCGCGGGCTCCTGGGCACCGCACCTGCTGAGC 3′, and the antisense (nt. 131-175), 5′ TGGGCGCCGGGCTGGCTGTCACCTCGCTCAGCAGGTGCGGTGCCC 3′.

[0075] Expression: The entire coding region of the human 5-HT_(4B) receptor (1338 bp), including 27 bp of 5′ untranslated (5′ UT) and 50 bp of 3′ untranslated sequence (3′ UT), was cloned into the SalI and EcoRI sites of the polylinker-modified eukaryotic expression vector pCEXV-3 (Miller et al, 1986), called EXJ.HR (unpublished data). The construct involved the ligation of three fragments from partial overlapping human placenta genomic and fetal brain cDNA clones: the start codon through TM 3 on a 0.5 kb NcoI-NcoI genomic fragment (the vector-derived SalI site was used for subcloning instead of the internal insert-derived NcoI site, at the 5′ end), TM 3 alone synthesized as overlapping oligonucleotides (based on previously determined cDNA sequence) with NcoI and KpnI termini, and TM 3/4 loop through the stop codon and 3′ UT, on a 0.8 kb KpnI-EcoRI cDNA fragment. Monkey kidney cells (Cos-7) were transiently transfected with plasmid hp78a/EXJ (expression vector encoding the human 5-HT_(4B) receptor gene) using DEAE dextran methodology (reagents obtained from Specialty Media, Lavellette, N.J.). Cells were crown as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island, N.Y.; 23) in a controlled environment (37° C., 5% CO₂). Stable cell lines were obtained by cotransfection with the plasmid hp78a/EXJ (expression vector containing the human 5-HT_(4B) receptor gene) and the plasmid pGCcos3neo (plasmid containing the amino glycoside transferase gene) into LM (tk⁻) cells, using calcium phosphate technique. The cells were grown, in a controlled environment (37° C., 5% CO₂), as monolayers in Dulbecco's modified Eagle's Medium (GIBCO, Grand Island, N.Y.) containing 25 mM glucose and supplemented with 10% bovine calf serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate. Stable clones were then selected for resistance to the antibiotic G-418 (1 mg/ml) as described previously (Weinshank et al., 1990) and membranes were harvested and assayed for their ability to bind [³H] hydroxytryptamine as described below (see Radioligand Binding Assays).

[0076] Macrolocalization (PCR/Tissue Transcriptional Expression Studies): Human tissues (NDRI) were homogenized and total RNA extracted using guanidine isothiocyanate/CsCl cushion method as previously described (Kingston, 1987). cDNA was prepared from 5 μg of total RNA with random hexanucleotide primers (500 pmoles) using Superscript reverse transcriptase (BRL) in 50 mM Tris-HCl, pH 8.3, buffer containing 40 U RNasin, 2.5 mM MgCl₂, 50 mM KCl and 1 mM dNTPs, at 42° C. for 1 hr. RNase H (2 U) was added, incubated for 20 min. at 37° C., followed by heating at 95° C. for 5 min. and chilled on ice. An aliquot of the first strand cDNA was diluted (1:5) in a 50 μl PCR reaction mixture (200 μM dNTPs final concentration) containing 1.25 U of Taq polymerase in the buffer supplied by the manufacturer (Cetus Corp.), and 1 μM of primers in a PCR protocol (the 5′ and 3′ oligos were the same as used for screening cDNA libraries; see above). The PCR amplification reaction was carried out by first a 5 min. incubation at 95° C. followed by 30 rounds of the following cycle: 2 min. at 94° C., 2 min. at 68° C., 3 min. at 72° C., followed at the end by 10 min. incubation at 72° C. In order to control for the amplification of DNA (carried over during the RNA extraction), control PCR reactions were run in parallel with RNA diluted in the same manner as the cDNA sample. The PCR products were run on a 1.5% agarose gel and transferred to charged nylon membrane (ZetaProbe, Bio-Rad). Filters were hybridized with end-labeled (with [γ-³²P]ATP) internal probe to the PCR primers (this oligo was the same as used for screening the initial human fetal brain cDNA library; see above), washed under high stringency (50° C.), and exposed at −70° C. to Kodak XAR film in the presence of an intensifying screen, as described above.

[0077] Microlocalization (In situ hybridization): Rats used for the in situ hybridization and receptor autoradiographic studies were euthanized with CO₂ and decapitated, and the brains dissected out and frozen in cold isopentane. The tissues were then mounted on cryostat chucks and sectioned serially at 11 μm for in situ hybridization, or at 20 μm for receptor autoradiography, in a cryostat maintained at −20° C. The sections were thaw-mounted on microscope slides coated with poly-1-lysine, air dried for one hour at room temperature, and stored at −80° C. until used.

[0078] Antisense and sense oligonucleotides (45mers) to the rat 5-HT_(4B) mRNA were synthesized on a Cyclone Plus DNA synthesizer (Milligen/Biosearch). Probes were 3′-end labeled with ³⁵S-dATP (1200 Ci/mmol, New England Nuclear, Boston, Mass.) to a specific activity of 10⁹ dpm/μg using terminal deoxynucleotidyl transferase (Boehringer Mannheim; Indianapolis, Ind.). The radiolabeled probes were purified on Biospin 6 chromatography columns (Bio-Rad; Richmond, Calif.), and diluted in hybridization buffer to a concentration of 1.5×10⁴ cpm/μl. The hybridization buffer consisted of 50% formamide, 4× sodium citrate buffer (SSC; 1×=0.15 M NaCl and 0.015 M sodium citrate), 1× Denhardt's solution (0.2% polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serum albumin), 50 mM dithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/ml yeast tRNA, and 10% dextran sulfate.

[0079] Tissue sections were warmed to room temperature for 1 hr prior to use. The sections were fixed in 4% (w/v) paraformaldehyde in 10 mM phosphate-buffered saline (PBS), washed twice in PBS, rinsed with 5 mM DTT in water, acetylated for 10 min with 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine, and rinsed twice in 2× SSC. The sections were then dehydrated in graded ethanols, delipidated with chloroform and air dried. One hundred μl of the diluted probe was applied to each section, which was then covered with a Parafilm coverslip. Hybridization was carried out overnight in humid chambers at 40 to 55° C. The following day the sections were washed in two changes of 2× SSC for one hour at room temperature, in 2× SSC for 30 min at 50-60° C., and finally in 0.1× SSC for 30 min at room temperature. Tissues were dehydrated in graded ethanols and apposed to Kodak XAR-5 film for 3 days to 2 weeks at −20° C., then dipped in Kodak NTB3 autoradiography emulsion diluted 1:1 with 0.2% glycerol water. After exposure at 4° C. for 2 to 4 weeks, the slides were developed in Kodak D-19 developer, fixed, and counterstained with hematoxylin and eosin.

[0080] Microlocalization (Receptor autoradiography) [³H]5-CT binding was performed as follows. Mounted tissue sections (20 μm) were brought to room temperature, and preincubated for 15 min in buffer containing 0.17 M Tris-HCl, 4.0 mM CaCl₂, 0.01% (w/v) ascorbic acid, and 10 μM pargyline. The slides were incubated in 0.5 nM [³H]5-carboxamidotyptamine (NEN, 50.4 Ci/mmol), for 1 hr at room temperature. To discriminate the 5-HT_(4B) receptor from other serotonin receptors which also bind [³H]5-CT, 100 nM PAPP and 160 nM (−)pindolol were used as masks. As the 5-HT_(4B) receptor has a very high affinity for methiothepin, 5 nM methiothepin was added to the radioligand to eliminate [³H]5-CT binding to the 5-HT_(4B) receptor. Nonspecific binding was determined by adding 1 μM ergotamine to the radioligand. Following incubation in the radioligand, the slides were washed twice for 20 min in the above buffer at 4° C., rinsed briefly in ice-cold water, and dried under a gentle stream of warm air. The slides were apposed to Hyperfilm (Amersham) at room temperature for 4-6 weeks, after which the films were developed with D-19 (Kodak), fixed, and air dried.

[0081] Membrane Preparation: Cos-7 cells transiently transfected with the human 5-HT_(4B) receptor gene were allowed to grow for 48 hrs. and membranes were harvested as previously described (Branchek et al., 1990). Membrane homogenates were kept on ice and utilized within one hr. for radioligand binding experiments. Protein concentrations were determined by the method of Bradford (Bradford, 1976) using bovine serum albumin as the standard.

[0082] Radioligand Dinding studies: Binding experiments were performed as previously described using [³H]5-HT as the radioligand (Zgombick et al., 1991). Eight concentrations of [³H]5-HT (1-100 nM) were used in saturation studies; seven concentrations of unlabelled compound and 5 nM [³H]5-HT were used in competition experiments. Unlabeled 5-HT (10 μM) was used to define nonspecific binding. Assays were terminated by vacuum filtration (Brandel, Gaithersburg, Md.) and the remaining radioactivity was quantified using a Beckman 500TA liquid scintillation counter (Beckman Instruments, Fullerton Calif.)

[0083] Measurement of cAMP Formation: Transiently transfected Cos-7 cells expressing the human 5-HT_(4B) receptor gene, mock-transfected Cos-7 cells, untransfected Cos-7 cells and stably transfected LM(tk−) cells were tested for the ability of 5-HT to modify intracellular cAMP levels. Both the 5-HT-mediated inhibition and stimulation of cAMP levels were evaluated in these three intact cell preparations. Intracellular cAMP formation was measured by radioimmunoassay (cAMP Radioimmunoassay kit, Advanced Magnetics, Cambridge, Mass.) using methodology outlined by Zgombick et al. (1991). Radioactivity was quantitatied using a Packard COBRA Auto Gamma Counter (equipped with data reduction software).

[0084] Data Analysis: Binding data was analyzed by nonlinear regression analysis (Accufit and Accucomp, Lundon Software, Chagrin Falls, Ohio). The Cheng-Prusoff equation was used to convert IC₅₀ values to K_(i) values. Functional data was fitted to a four parameter logistic equation to obtain response parameters (EC₅₀, E_(max), nH; Inplot, GraphPad, San Diego, Calif.). All experiments were performed a minimum of three times.

[0085] Drugs: Drugs were obtained from the following companies; [³H]5-HT specific activity=20.4-28.0 Ci/mmole New England Nuclear, Boston, Mass.); 5-HT, ergotamine, ergonovine, oxymetazoline, (±) -pindolol, 5′-guanylylimidodiphosphate (Sigma, St. Louis, Mo.); 5-CT, DP-5-CT, 5-MeOT, 5-MeO-DMT, (±)-α-Me-5-HT, 2-Me-5-HT, tryptamine, DPAT, DOI, ketanserin, methysergide, 1-naphthylpiperazine, PAPP, spiperone, TFMPP, yohimbine, zacopride (Research Biochemical Inc, Natick, Mass.); lysergol, methylergonovine (Aldrich Chemicals, Milwaukee, Wis.) rauwolscine (Accurate Chemicals, Westbury, N.Y.); methiothepin (Biomol Research Laboratories, Plymouth Meeting, Pa.). All other chemicals were the highest purity available commercially.

RESULTS

[0086] We screened a human genomic placenta library, under medium stringency conditions, with oligonucleotide probes directed to the third, fifth, sixth and seventh transmembrane regions of the Drosophila serotonin receptor gene, Dro5HTR (Witz et al, 1990). A total of 3 positive clones were isolated and characterized by Southern blot analysis. Two clones were identical and the other clone was an overlapping fragment of the other two. One of the two identical clones, designated hp78a, contained a 1.8 kb EcoRI/PstI fragment which hybridized with the Drosophila-derived oligonucleotide probes and was subsequently subcloned into a pUC vector. DNA sequence analysis indicated greatest homology to the Drosophila SHT receptor, which is coupled to adenylate cyclase stimulation (Witz et al, 1990). Hydrophobicity plot of this subclone demonstrated regions of hydrophobic amino acid residues flanking segments of hydrophilic amino acid residues, consistent with it being a member of the seven transmembrane G-protein coupled receptor gene superfamily (Savarese et al., 1992). In addition, this clone contained an alanine residue in the predicted fifth transmembrane region, consistent with it being a member of the serotonin subfamily; this alanine residue distinguishes the serotonin subfamily from other catecholamine receptors (which have a serine residue at this position; Weinshank et al., 1992b). This clone encoded TM4 through the carboxyl terminus, including an intron upstream of the predicted second intracellular loop.

[0087] In order to obtain a full-length clone, aliquots of human cDNA libraries totaling ≈1.5×10⁶ recombinants were screened by polymerase chain reaction using specific oligonucleotide probes from sequence determined off the genomic clone (see Materials and Methods). A positive-containing human fetal brain cDNA library (stratagene, LaJolla, Calif.) in λ ZapII (≈1.5×10⁶ recombinants) was screened using traditional plaque hybridization with an internal probe (see Materials and Methods) and resulted in the isolation of one positive partial-length cDNA clone, hFB9a, which contained TM3 through the stop codon and overlapped with the original genomic clone, hp78a (the intron between Tm3 and Tm4 was absent due to mRNA splicing). Because this cDNA clone was not full-length, the same human fetal brain cDNA library was screened with a probe derived from the most upstream non-conserved region of the hFB9a cDNA clone, corresponding to Tm3/4 loop (see Materials and Methods). This screen resulted in the isolation of another positive but partial-length cDNA clone, hFB44a, which contained only TM2 through TM3 and overlapped with the hFB9a cDNA clone within TM3. Again, to obtain 5′ cDNA clones, the human fetal brain cDNA library was screened with a probe derived from the most upstream non-conserved region of the hFB44a cDNA clone, corresponding to TM2/3 loop (see Materials and Methods). This screen resulted in the isolation of another positive but partial-length cDNA clone, hFB41a, which contained part of the amino terminus through TM3, but missing about 20 amino acids from the start methionine residue (similarly, this clone overlapped with hFB44a cDNA clone in TM2 through TM3). Finally, to obtain the complete amino terminus, a human genomic placenta library was screened under high stringency with a probe derived from the non-conserved amino terminus region of the hFB41a cDNA clone. A total of 2 different positive clones were isolated from this genomic screen and were characterized by Southern blot analysis. One clone represented a pseudogene of the 5-HT_(4B) receptor (unpublished data), whereas the other genomic clone contained the initiating methionine residue through the intron between TM3 and TM4. A 0.5 kb NcoI/NcoI fragment containing the initiating methionine to the NcoI site within TM3 was subcloned in pGEM.

[0088] The complete full-length gene was constructed in two parts: the first part involved ligating two fragments from two cDNA clones and a synthetic double-stranded oligonucleotide into pBluescript, and the second part involved ligating the amino terminus-containing genomic fragment and a fragment from the first part construct into an expression vector. The first part of the construction involved ligating a 0.4 kb SalI/NcoI fragment from hFB41a cDNA clone (SalI site is vector-derived, whereas the NcoI site is in Tm3), an annealed complementary double-stranded oligonucleotide fragment with designed NcoI and KpnI termini (≈100 bp; sequence based on data from the hFB9a, hFB44a, and hFB41a cDNA clones) and a 0.8 kb KpnI/EcoRI fragment from hFB9a cDNA clone (EcoRI is vector-derived, whereas the KpnI site is in TM3/4 loop) into pBluescript digested with SalI and EcoRI. The second part of the construction was perfromed by ligating the 0.5 kb SalI/NcoI genomic subclone, containing the initiating methionine through TM3 (SalI is vector-derived, whereas the NcoI is within TM3; this fragment was obtained by partial digestion with Nco I and complete digestion with SalI), with a 0.9 kb NcoI/EcoRI synthetic oligonucleotide/cDNA fragment from the first part construct (containing TM3 through the stop codon) into the expression vector digested with SalI and EcoRI.

[0089] The genomic/cDNA full-length construct in the expression vector (called hp78a/EXJ) contains an open reading frame of 1335 bp (with 27 bp at 5′ UT and 50 bp at 3′ UT) and encoding a protein of 445 aa in length, having a relative molecular mass of ≈49,000 daltons.

[0090] Hydropathy analysis of the protein is consistent with a putative topography of seven transmembrane domains, indicative of the G protein-coupled receptor family. Initial sequence analysis revealed that clone hp78a/EXJ was most related to a serotonin receptor since it contained a number of conserved structural features/residues found among the members of the serotonin receptor family, including conserved aspartic acid residues in TM2 and TM3, the Asp-Arg-Tyr sequence at the end of TM3, the conserved alanine residue in the fifth transmembrane region, and the conserved proline residues of Tm 4-7 (Hartig 1989; Hartig et al., 1990). Other features of this human 5-HT_(4B) receptor gene are the presence of two potential sites for N-linked glycosylation in the amino terminus (asparagine residues 5 and 66; FIG. 1) and the presence of several serines and threonines in the carboxyl terminus and intracellular loops, which may serve as sites for potential phosphorylation by protein kinases.

[0091] Human Tissue Distribution of RNA Coding for 5-HT_(4B) Receptor Gene. Total RNA isolated from various human tissues were converted to single-stranded cDNA by random-priming with reverse transcriptase. cDNAs were amplified by PCR using human 5-HT_(4B) receptor gene specific PCR primers. PCR products were run on a 1.5% agarose gel, blotted onto nylon membranes and hybridized to internal gene-specific oligonucleotides. Following hybridization, blots were washed under high stringency. Positive control consisted of gene-specific recombinant plasmid; dH₂O served as a negative control, containing all reagents except template cDNA, RNA or plasmid. PCR amplification and Southern blotting of RNA samples not treated with reverse transcriptase were negative. TABLE 1 Human tissue localization of 5-HT_(4B) receptor mRNA HUMAN TISSUES 5-HT_(4B) mRNA Prostate III + Prostate IV + Prostate V + ½ Testes +++ Bladder +++ Endometrium (uterus) ½ + Myometrium (uterus) ½ + Atrium ½ + Mesentery + Nasal Mucosa ++ Penis ++ Skin (−) Tongue (−)

[0092] The expression of hp78a transcripts was analyzed by amplification of cDNA derived from RNA isolated from various human tissues (reverse transcription PCR or RT-PCR) (FIG. 3). We were able to examine, selectively, the tissue distribution of the functional hp78a clone (and not the pseudogene) by using gene-specific PCR primers and gene-specific internal oligonucleotide probes, which failed to detect the pseudogene (data not shown). We demonstrated comparable quantities of starting RNA for all tissues by conducting control RT-PCR with primers for the moderately high level constituitively expressed genes, actin and glyceraldehyde 3-phosphate dehydrogenase (Clontech; data not shown). The mRNA encoding the 5-HT_(4B) receptor (hp78a) receptor is expressed at high levels in the brain and at low levels in a variety of peripheral tissues (kidney, liver, pancreas, prostate, uterus, mesentary and spleen). Based on the pharmacological profile of the 5-HT_(4B) receptor and its relationship to the previously characterized 5-HT receptors which mediate smooth muscle relaxation, we tested a number of additional tissues for RNA expression. Interestingly, we detected high levels of mRNA for 5-HT_(4B) in the coronary artery and in various regions of the gastrointestinal tract, including the stomach, bladder, descending colon and ileum, consistent with the possible role of 5-HT_(4B) as a smooth muscle relaxant receptor (FIG. 3).

[0093] The results of the in situ hybridization studies (Table 2) indicated that the distribution of the 5-HT_(4B) mRNA overlapped that of the 5-HT_(4B) receptor in some areas. The most intense hybridization signals were observed over neurons in the thalamus, the anterior hippocampal rudiment, and over CA3 in the hippocampus. Other regions containing hybridization signals included the septum, the hypothalamus, the centromedial amygdala, and the periaquaductal gray. Interestingly, two areas which exhibited a robust receptor binding signal, the globus pallidus and the substantia nigra, contained no 5-HT_(4B) hybridization signals, suggesting a presynaptic localization for 5-HT_(4B) receptors in these two related structures.

[0094] Specific binding of [³H]5-CT was observed in many areas of the rat brain. Following incubation with 1 μM ergotamine, this binding was completely eliminated. Addition of PAPP and (−)pindolol to the radioligand markedly reduced the binding in a number of regions, most notably in the deep layers of the cerebral cortex, hippocampus, dorsal raphe, and spinal cord. Areas in which [³H]5-CT binding remained following addition of the masks were layers 1-3 of cortex, septum, globus pallidus, thalamus, hypothalamus, centromedial amygdala, substantia nigra, periaquaductal gray, and superior colliculus. The radioligand binding in all of these areas was abolished by the addition of 5 μM methiothepin to the incubation regimen, indicating that the observed binding was attributable to 5-HT_(4B) receptors. TABLE 2 Localization of the 5HT_(4B) Receptor/mRNA in Rat Brain [³H]5-CT Binding 5-HT_(4B) REGION Sites mRNA Cerebral cortex + + layers I, II and III Septum + + Globus pallidus + − Bed n. stria terminalis + + Thalamic nuclei + + Hypothalamic n. + + Amygdala (centromedial + + complex) Hippocampus^(A) + + Central gray + + Substantia nigra + − Superior colliculus + ± Dorsal cortex Inferior + nd Colliculus Cerebellum − +

[0095] Monkey kidney cells transiently expressing the gene encoding the novel human 5-HT receptor were used for pharmacological evaluation. Membranes harvested from transiently transfected Cos-7 cells exhibited high affinity, saturable [³H]5-HT binding. Nonlinear analysis of [³H]5-HT saturation data yielded an equilibrium dissociation constant (K_(d)) of 8.5±0.8 nM and a binding site density (B_(max)) of 6.6±0.8 pmol/mg protein. Specific [³H]5-HT binding was greater than 90% of total binding at a radioligand concentration equal to the K_(d) value. High affinity [³H]5-HT binding to membranes prepared from transient transfectants was reduced significantly (75%) by 100 μM Gpp(NH)p, a nonhydrolyzable analog of GTP. Untransfected host cells did not displayed specific [³H]5-HT binding.

[0096] To further assess the pharmacological identity of the newly isolated serotonin receptor gene, detailed binding properties of clone hp78a were determined from nonlinear analysis of competition of high affinity [³H]5-HT binding. Specific [³H]5-HT binding was completely displaced in a monophasic manner (n_(H)=1) by a variety of structurally diverse serotonergic ligands. The rank order of potency of these compounds to displace specific [3H]5-HT binding was 5-CT>methiothepin>Metergoline=DHE=5-MeOT>5-HT>2-Br-LSD>DP-5-CT>DPAT>sumatriptan>ICS 203930 (Tables 3 and 4). Several ergot derivatives exhibited high affinity (K_(i)<20 nM) for clone 5-HT_(4B) receptor and includemetergoline, dihydroergotamine, ergotamine, and mesulergine. Serotonergic ligands which display subtype selectivity and which exhibit low affinity (K_(i)>100 nM) for the clone hp78a include DPAT (5-HT_(1A)), sumatriptan (5-HT_(1D)), ketanserin (5-HT₂), and zacopride (5-HT₃). The substituted benzamides (ICS 203930, MDL 29429, zacopride), compounds which are active at the functional 5-HT₄ receptor, also displayed low affinity (K_(i)>500 nM) for the 5-HT_(4B) receptor. Other biogenic amines did not show appreciable affinity (K_(i)>1 μM) for clone hp78a (Tables 3 and 4). TABLE 3 Apparent dissociation constants (K_(i) values) of serotonergic ligands for clone hp78a. Membranes were incubated with 5 nM [³H]5-HT in the presence of seven concentrations (1000-fold concentration range) of unlabelled competitors for 30 min at 37° C. Nonspecific binding was defined by 10 μM unlabelled 5- HT. Affinity constants (K_(i) values) were calculated from IC₅₀ values by nonlinear regression analysis using the Cheng-Prusoff equation. K_(i) values are expressed as mean values ± S.E.M. from 2-5 determinations. COMPOUND K_(i) (nM) 5-Carboxamidotryptamine 0.93 ± 0.20 Methiothepin 3.7 ± 1.0 5-Methoxytryptamine 5.1 ± 0.4 Metrogoline 6.4 ± 1.4 Dihydroergotamine 6.9 ± 0.9 5-Hydroxytryptamine 8.1 ± 1.3 Ergotamine  15 Mesulergine 18 ± 5  Bufotenin  26 2-Bromo-LSD 30 ± 10 Dipropyl-5- 37 ± 21 carboxamidotryptamine 5-Methoxy-N,N-dimethyl  43 tryptamine Ritanserin 45 ± 10 Clozapine 78 ± 10 Methysergide 83 ± 5  1-Naphthylpiperazine 83 ± 19 Spiperone 110 ± 17  Cyproheptadine 123 ± 16  Bromocriptine 137 ± 2  Tryptamine 149 ± 20  m-Chlorophenylpiperazine 312 8-Hydroxy-N,N-dipropyl- 466 ± 46  aminotetralin α-Methyl-5-hydroxy- 509 tryptamine 5-Benzyloxytryptamine 729 Sumatriptan 951 ± 118 Ketanerin 1334 ± 241  Yohimbine 2850 ± 354  2-Methyl-5-hydroxy- 3400  tryptamine Pindolol >5000  Zacopride >5000 

[0097] TABLE 4 Percent displacement of specific [³H]5-HT binding to membranes derived from cells transiently expressing the hp78a gene by serotonergic ligands. Membranes were incubated with 5 nM [³H]5-HT and one concentration of unlabelled competitor for 30 min at 37° C. Nonspecific binding was defined by 10 μM unlabelled 5-HT. CONCENTRATION PERCENT COMPOUND TESTED INHIBITION d-LSD  30 nM 77 BRL 29429  100 nM 8 ICS 203930 1000 nM 10 Spiroxatrine  30 nM 12 Propanolol  100 nM 0 PAPP  100 nM 21 CGS 12660B  100 nM 15 Quipazine  100 nM 3 Rauwolscine  100 nM 8 DOI  100 nM 20 Oxymetazoline  10 nM 6 Dopamine  30 nM 0 Norepinephrine  30 nM 0 Histamine  100 nM 10

[0098] The ability of clone hp78a to functionally couple to adenylate cyclase was tested using intact Cos-7 cells transiently expressing the gene encoding hp78a. Both the stimulation of basal cAMP release and inhibition of FSK-stimulated cAMP response were investigated. 5-HT (1 μM) had no effect on either basal or FSK-stimulated adenylate cyclase activity in untransfected or mock-transfected Cos-7 cells (data not shown), indicating that endogenous cyclase-coupled serotonin receptors are not expressed in untransfected cells. Addition of 5-HT (1 μM) to transfected Cos-7 cells elicited a 7-fold stimulation of basal cAMP release (0.035±0.004 pmol/ml/10 min); inhibition of either the basal or FSK-stimulated cAMP release was not observed. Coincubation of 1 μM FSK and 1 μM 5-HT evoked a synergistic cAMP response, resulting in a 75-fold stimulation of intracellular cAMP accumulation (data not shown). 5-CT and 5-MeOT were also potent agonists at 1 μM, interestingly 5-CT appeared to be more potent than 5-HT itself, whereas 8-OH-DPAT stimulated the basal cAMP response by only about 2 fold at 1 μM. Methysergide, metergoline and methiothepin at 10 μM, antagonized the 5-HT response almost completely, whereas ICS-205-930 had a weak inhibitory effect at 100 μM. Full dose-response curves were determined for 5-HT and 5-CT. 5-HT exhibited an EC₅₀ value of 992±345 nM and a maximum stimulation (E_(max) value) of 2,191±715% basal cAMP release (n=4) whereas 5-CT had a higher affinity (EC₅₀=75±15 nM) with a similar E_(max) (2029±47% basal cAMP release, n=4) (Table 5; FIGS. 4 and 5). TABLE 5 Pharmocological profile for the cAMP response using the human 5-HT_(4B) receptor transiently expressed in Cos-7 cells. cAMP measurements on intact cells were as described under Methods and Materials. Drug activity was measured in triplicate at the single indicated. Results for agonists are expressed as fold cAMP stimulation relative to basal levels (basal, 0.053 ± pmol/ml/10 min). Data for antagonists are expressed as % inhibition of the cAMP response to 1 μM 5-HT. FOLD % INHIBITION STRUCTURAL cAMP STIM- of 5-HT CLASS DRUG [DRUG] ULATION RESPONSE INDOLEAMINES 5-HT  1 μM  6.7 ± 0.65 5-CT  1 μM  28 ± 5.5 5-MeOT  1 μM  6.0 ± 0.08 ICS-205-930 100 μM 0  36 ± 3.0 ERGOLINES Methysergide  10 μM  1.7 ± 0.43  82 ± 1.5 Metergoline  10 μM 0  96 ± 1.0 PIPERAZINES Methiothepin  10 μM 0 100 ± 0  2,Bromo LSD  1 μM 0  95 ± 4.5 BENZAMIDES BRL 29429  10 μM 0 AMINOTETRALIN DPAT  1 μM 1.7 ± 0.4

[0099] In stably transfected LM (tk⁻) cells 5-HT produced a maximum stimulation of 400% of basal cAMP accumulation. Inhibition of either the basal or FSK-stimulated cAMP release was not observed. The affinity values of various compounds tested are summarized in Table 6. In general the EC₅₀ values of agonists obtained from functional studies were about one order of magnitude lower than the K_(i) values obtained from the binding assays. 5-CT and 5-MeOT were potent agonists having comparable intrinsic activity to 5-HT_(4B) however 5-CT had significantly greater affinity (approximately 10-fold) than 5-HT. 8-OH-DPAT was a weak agonist producing approximately half of the maximum response elicited by 5-HT. All the ergot alkaloids tested behaved as silent antagonists with K_(B) values not deviating significantly from K_(i) values obtained from binding assays. Methiothepin was also a potent silent antagonist (Table 6). TABLE 6 Pharmacological profile for the cAMP response using the human 5-HT_(4B) receptor stably expressed in LM(tk-) cells. cAMP measurements on intact cells were as described under Methods and Materials. Average maximum response to 5-HT in these cells is 400% stimulation of basal cAMP production. E_(max) values are normalized to the 5-HT maximal response. AGONIST indictaes that the maximal response to the drug was at least 80% of the maximum response obtained with 5-HT (400% stimulation of basal cAMP accumulation). Results are means ± S.E.M. of at least 3 experiments performed in triplicate. Dissociation constant of antogonist (K_(B)) was calculated According to the formula: K_(B) = [B]/(A′/A) − 1], where [B] is the concentration of antagonist, A′ and A the EC₅₀ values of agonist measured respectively in the presence and in the absence of antagonist (Furchgott, 1972). E_(max) (% 5-HT K_(B) INTRINSIC DRUG EC₅₀ (nM) RESPONSE) (nM) ACTIVITY 5-CT 17 ± 2  110 ± 19  AGONIST 5-MeO-T 173 ± 1  100 ± 5  AGONIST 5-HT 189 ± 17  100 AGONIST DP-5-CT 2,398 ± 49   86 ± 12 AGONIST 8-OH-DPAT 17,160 ± 1,254  51 ± 14 PARTIAL AGONIST d-LSD  0 2.7 ± 0.5 ANTAGONIST Methiothepin  0 11 ± 1  ANTAGONIST 2-Br-LSD  0 12 ± 3  ANTAGONIST DHE  0 20 ± 4  ANTAGONIST Mesulergine  0 20 ± 1  ANTAGONIST Bromocriptine  0 240 ± 10  ANTAGONIST

DISCUSSION

[0100] We have cloned DNA representing a novel human serotonin receptor (hp78a or 5HT_(4B)) from human brain cDNA and genomic DNA. Of all known G protein-coupled receptor sequences (EMBL/Genbank Data Base), the greatest homology was between clone hp78a receptor gene and the Drosophila 5HTR serotonin receptor gene (Witz et al., 1990). Comparison of clone hp78a deduced amino acid sequence with known G protein-coupled receptor sequences indicates the greatest concentration of identical amino acids to be in the transmembrane domains. In these TM regions, the percentage of identity for hp78a clone is 57% compared to Dro5HTR1, 39-53% with serotonin receptor subfamily (see FIG. 2), 40-50% with adrenergic receptor subfamily, 44-49% with the dopamine receptor subfamily, 37-41% with histamine receptor subfamily, 27-28% with peptide receptors and 32-33% with adensine receptors. Both the alignment and percent identity of this human hp78a sequence, relative to other G protein-coupled receptors or other members of the serotonergic subfamily, strongly suggest that this is a new receptor. Because the TM homology between the clone hp78a receptor and any of the known serotonin receptors is below 55% and the fact that this receptor is positively-coupled to adenylate cyclase (see RESULTS and below), we are naming this clone 5HT_(4B).

[0101] Localization of transcripts for this receptor indicate a relatively broad tissue distribution. Of the tissues examined, the human 5-HT4B (clone hp78a) receptor mRNA was detected in greatest abundance in the brain, testes and bladder, with moderate to low levels found in other peripheral tissues, with no signal detected in skin and tongue.

[0102] The distribution of the 5-HT_(4B) receptor and its mRNA, described herein, indicates that this novel serotonin receptor may subserve functions within several brain systems. The limbic system (septum, centromedial amygdala, anterior hippocampal rudiment, hypothalamus) is particularly well-represented, suggesting a role for the 5-HT_(4B) receptor in affective processes. The finding that this receptor binds clozapine very well supports this notion, intimating that the 5-HT_(4B) receptor may be involved in schizophrenia or in other neurologically based affective disorders. In addition, the localization of the clone hp78a mRNA to thalamic nuclei which project to neocortex suggests that this receptor is involved in sensory processes. The localization in the raphe nuclei indicate possible function as autoreceptors which can be targeted as novel therapies through 5-HT_(4B) function in anxiolytics or antidepressants. Since the distribution of 5-HT receptors is generally preserved across species, the localization results from non-human species, such as rat or guinea pig, are considered most likely analogous to human brain.

[0103] The binding profile obtained with the clone hp78a receptor appears to represent a distinct serotonin receptor subtype based upon its unique pharmacological profile. The rank order of compounds to displace [³H]5-HT binding (Table 3) does not match the binding properties obtained with other [³H]5-HT labeled binding sites (Xiong and Nelson, 1989; Zemlin et al., 1991) or pharmacological properties of pharmacologically defined serotonin receptors described in a variety of functional preparations (see below). The high affinity of 5-CT (K_(i)=0.5 nM) and low affinity of sumatriptan (K_(i)>750 nM) for the clone hp78a receptor matches the binding properties of a [³H]5-CT binding site recently identified in guinea pig striatal homogenates (Mahle et al., 1992). However, a direct comparison of the pharmacological properties of the clone hp78a receptor to that of the [³H]5-CT binding site cannot be made since a complete characterization of the binding site is lacking and no functional response has been demonstrated.

[0104] Binding criteria used to classify 5-HT₁ receptor subtypes include high affinity for 5-HT and 5-CT. These two compounds exhibited high affinity for the clone hp78a receptor (Table 3). However, both the recently cloned human 5-HT_(1E) (McAllister et al., 1992; Zgombick et al., 1992) and 5-HT_(1F) (Adham et al., 1992) receptors display high affinity for 5-HT (K_(i)<10 nM) and low affinity for 5-CT (K_(i)>700 nM). The 5-HT_(1E) and 5-HT_(1F) receptors are members of the 5-HT₁ receptor family based upon their TM homologies to that of other cloned members of this receptor family (5-HT_(1A), 5-HT_(1B), 5-HT_(1D α), 5-HT_(1Dβ)) and second messenger coupling (inhibition of adenylate cyclase). Moreover, while the cloned 5-HT_(1C) receptor binds [³H]5-HT with high affinity, it is considered a 5-HT₂ receptor based upon it high TM homology (75%) to the cloned 5-HT₂ receptor and functional coupling to phospholipase C (Hartig et al., 1990; Julius, 1992). Based upon these observations, it is difficult to classify clone hp78a receptor as a 5-HT₁ receptor subtype based solely on its radioligand binding properties.

[0105] Functional responses of the clone hp78a receptor provide additional insight into its identity. The clone hp78a receptor gene transiently expressed in Cos-7 cells coupled to stimulation of adenylate cyclase. Both 5-HT and 5-CT elicited a 20-fold increase in cAMP release over basal levels. The K_(i) values 5-HT and 5-CT obtained in binding assays were approximately 100-fold higher affinity than the EC₅₀ values of these agonists obtained from functional assays. The rank order of potency of agonists observed in the functional assay 5-CT>5-HT≧5-MeOT>8-OH-DPAT is very similar to the rank order of these compounds to displace specific [³H]5-HT binding. Stable LM(tk⁻) cells expressing the 5-HT_(4B) receptor gave very similar pharmacology to the transiently transfected COS-7 cells with agonists producing essentially identical rank order of potencies, however the maximum cAMP response produced by agonists was lower in LM(tk⁻) cells. Antagonists had the following rank order of potencies: d-LSD>methiothepin≧2-Br-LSD>mesulergine=DHE.

[0106] The ability of serotonin to activate adenylate cyclase has been demonstrated in several systems including: embryonic mouse colliculi (5-HT₄, Dumius et al., 1988), horse brain glial membranes (5-HT₁-like, Fillion et al., 1980), rat and guinea-pig hippocampal membranes (5-HT_(1A), Shenker et al., 1987), human atria (5-HT₄, Kaumann et al., 1989), NCB20 neuroblastoma hybrid cells (5-HT₄-like, Conner and Mansour, 1990; Cossery et al., 1990) and neonatal porcine vena cava (5-HT₁-like, Trevethick et al., 1984). The receptor encoded by HT_(4B) gene is distinct from the 5-HT₄ receptor identified in these preparations, although there are some common properties. Both the 5-HT₄ receptors found in the colliculi, atria and on the NCB20 cells and clone hp78a receptor have equipotency for 5-HT and 5-MeOT which stimulate cAMP release with low affinity. In addition, they are both relatively insensitive to 8-OH-DPAT, spiperone, ketanserin, and pindolol. However, methiothepin is a potent antagonist of the functional responses mediated by clone hp78a receptor but not of the colliculi and atrial 5-HT₄ receptor responses. Most critically, compounds used to define the “classical” 5-HT₄ receptor (agonists: cisapride, zacopride, BRL 29429; weak antagonist: ICS-205-930) are all either poorly active or inactive at the clone hp78a receptor.

[0107] A somewhat closer pharmacological relationship exists between the clone hp78a receptor and the 5-HT4-like receptor on NCB20 cells, including the potent antagonist activity of methiothepin. However, they are distinct entities based on the following differences: a) [³H]-5-HT binds with high affinity (Kd=8.5 nM) to clone hp78a receptor but it has a very low affinity (−200 nM) at the 5-HT receptors on NCB20 cells (Berry-Kravis and Dawson, 1983), b) the rank order of potency of agonists at hp78a is 5-CT>5-HT≧5-MeoT, whereas on NCB20 cells the order is 5-HT≧5-MeOT>5-CT (Conner and Mansour, 1990). In addition, clone hp78a receptor share several properties with the 5-HT receptor present in glial membranes (Fillion et al., 1980) These include: (a) the equilibrium dissociation constant of [³H]-5-HT K_(d)=10 Nm; (b) the EC₅₀ value for 5-HT stimulation of cAMP (500-1000 nM, and (c) Potent antagonism by: 2-bromoLSD, methysergide and methiothepin.

[0108] Criteria used to classify serctonin receptors into four distinct classes designated 5-HT₁, 5-HT₂, 5-HT₃, and 5-HT₄ is based upon binding properties, signal transduction mechanisms, and deduced amino acid sequences. According to the serotonin receptor classification scheme proposed by Bradley et al., (1986), clone hp78a receptor could be considered a member of the 5-HT₁ receptor family since 5-CT behaves as an agonist and this response is potently antagonized by the nonselective 5-HT₁ antagonist methiothepin. However, the positive coupling of clone hp78a receptor to adenylate cyclase seen here is not consistent with that reported for cloned members of 5-HT₁ receptor subfamily which couple to the inhibition of FSK-stimulation cAMP release and include the 5-HT_(1A) (Kobilka et al., 1987; Fargin et al., 1988), 5-HT_(1B) (Adham et al., 1992; Maroteaux et al., 1992), 5-HT_(1Dα) (Hamblin and Metcalf, 1991; Weinshank et al., 1992); 5-HT_(1Dβ) (Demchyshyn et al., 1992; Jin et al., 1992: Weinshank et al., 1992); 5-HT_(1E) (Levy et al., 1992 ; McAllister et al., 1992; Zgombick et al., 1992); and 5-HT_(1F) (Adham et al., submitted).

[0109] Although a useful construct, classification of serotonin receptors based solely on binding properties can be misleading. For example, the 5-HT_(1C) receptor, named originally by binding criteria, ([³H]5-HT binds with high affinity to the this subtype) is now considered a 5-HT₂ msubtype based upon the high degree (75%) homology within the TM domains to the 5-HT₂ receptor and second messenger coupling (phosphoinositide hydrolysis) (Hartig et al., 1990; Julius, 1992). Similarly, although the clone hp78a receptor binds [³H]5-HT with high affinity, its positive coupling to adenylate cyclase and the lower TM homology (˜50%) to other cloned members of the 5-HT₁ receptor family would seem to exclude the possibility clone hp78a encodes a 5-HT₁ subtype. Thus, it appears that TM homology comparisons and second messenger coupling are more accurate predictors for receptor classification than radioligand binding properties. Based upon these analyses, we propose that clone hp78a be designated the first member of the 5-HT₄ receptor subfamily. The pharmacological profile of clone hp78a is distinct from that of the pharmacologically defined 5-HT₄ receptor. Therefore, clone hp78a will be termed 5-HT_(4B), reserving the 5-HT_(4A) designation for a clone which will display the pharmacological profile of the 5-HT₄ receptor described in a variety of isolated tissue preparations (Bokaert et al, 1992).

[0110] In conclusion, the primary structure of the hp78a gene, as well as its unique pharmacological profile and positive coupling to adenylate cyclase obtained from transiently transfected cells, indicate that this gene may encode the first member of the 5-HT₄ receptor family. Clone hp78a is proposed to be designated as the 5-HT_(4B) receptor subtype since it does not display the pharmacological properties of the pharmacologically defined 5-HT₄ receptor. Additional cloning efforts will be required to isolate additional members of this newly recognized serotonin receptor family (Bockaert et al., 1992). Comparison of the pharmacological relationship of 5-HT_(4B) with serotonin receptors defined in tissue models indicates a possible identity with a collection of related receptors described in the vasculature. Several of these receptors appear to underlie relaxant responses in isolated blood vessels indicating potential therapeutic benefit in angina, coronary artery disease, atherosclerosis, and possibly cerebral blood vessel disorders leading to stroke. The presence of this subtype in the CNS also indicates potential use in disorders of higher cognitive processes as well as control of autonomic function. Moreover, the relatively high affinity (K_(i)=78 nM) of the atypical antipsychotic clozapine for the 5-HT₇ receptor, as well as the localization of this subtype in limbic and cortical regions, indicates a potential role of the 5-HT₇ subtype in neuropsychiatric disorders such as schizophrenia which involve serotonergic neurotransmission.

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1 17 1 1406 DNA Homo Sapiens 1 ccatgggcag cggcacacgg cggcgcgatg atggacgtta acagcagcgg ccgcccggac 60 ctctacgggc acctccgctc tttccttctg ccagaagtgg ggcgcgggct gcccgacttg 120 agccccgacg gtggcgccga cccggtcgcg ggctcctggg cgccgcacct gctgagcgag 180 gtgacagcca gcccggcgcc cacctgggac gcgcccccgg acaatgcctc cggctgtggg 240 gaacagatca actacggcag agtcgagaaa gttgtgatcg gctccatcct gacgctcatc 300 acgctgctga cgatcgcggg caactgcctg gtggtgatct ccgtgtgctt cgtcaagaag 360 ctccgccagc cctccaacta cctgatcgtg tccctggcgc tggccgacct ctcggtggct 420 gtggcggtca tgcccttcgt cagcgtcacc gacctcatcg ggggcaagtg gatctttgga 480 cactttttct gtaatgtctt catcgccatg gacgtcatgt gctgcacggc ctcgatcatg 540 accctgtgcg tgatcagcat tgacaggtac cttgggatca caaggcccct cacataccct 600 gtgaggcaga atgggaaatg catggcgaag atgattctct ccgtctggct tctctccgcc 660 tccatcacct tacctccact ctttggatgg gctcagaatg taaatgatga taaggtgtgc 720 ttgatcagcc aggactttgg ctatacgatt tactctaccg cagtggcatt ttatatcccc 780 atgtccgtca tgcttttcat gtactaccag atttacaagg ctgccaggaa gagtgctgcc 840 aaacacaagt ttcctggctt ccctcgagtg gagccagaca gcgtcatcgc cctgaatggc 900 atagtgaagc tccagaagga ggtggaagag tgtgcaaacc tttcgagact cctcaagcat 960 gaaaggaaaa acatctccat ctttaagcga gaacagaaag cagccaccac cctggggatc 1020 atcgtcgggg cctttaccgt gtgctggctg ccatttttcc tcctctcgac agccagaccc 1080 ttcatctgtg gcacttcctg cagctgcatc ccactgtggg tggagaggac atttctgtgg 1140 ctaggctatg caaactctct cattaaccct tttatatatg ccttcttcaa ccgggacctg 1200 aggaccacct atcgcagcct gctccagtgc cagtaccgga atatcaaccg gaagctctca 1260 gctgcaggca tgcatgaagc cctgaagctt gctgagaggc cagagagacc tgagtttgtg 1320 ctacaaaatg ctgactactg tagaaaaaaa ggtcatgatt catgattgaa agcagaacaa 1380 tggagaggaa ttcgatatca agctta 1406 2 445 PRT Homo Sapiens 2 Met Met Asp Val Asn Ser Ser Gly Arg Pro Asp Leu Tyr Gly His Leu 1 5 10 15 Arg Ser Phe Leu Leu Pro Glu Val Gly Arg Gly Leu Pro Asp Leu Ser 20 25 30 Pro Asp Gly Gly Ala Asp Pro Val Ala Gly Ser Trp Ala Pro His Leu 35 40 45 Leu Ser Glu Val Thr Ala Ser Pro Ala Pro Thr Trp Asp Ala Pro Pro 50 55 60 Asp Asn Ala Ser Gly Cys Gly Glu Gln Ile Asn Tyr Gly Arg Val Glu 65 70 75 80 Lys Val Val Ile Gly Ser Ile Leu Thr Leu Ile Thr Leu Leu Thr Ile 85 90 95 Ala Gly Asn Cys Leu Val Val Ile Ser Val Cys Phe Val Lys Lys Leu 100 105 110 Arg Gln Pro Ser Asn Tyr Leu Ile Val Ser Leu Ala Leu Ala Asp Leu 115 120 125 Ser Val Ala Val Ala Val Met Pro Phe Val Ser Val Thr Asp Leu Ile 130 135 140 Gly Gly Lys Trp Ile Phe Gly His Phe Phe Cys Asn Val Phe Ile Ala 145 150 155 160 Met Asp Val Met Cys Cys Thr Ala Ser Ile Met Thr Leu Cys Val Ile 165 170 175 Ser Ile Asp Arg Tyr Leu Gly Ile Thr Arg Pro Leu Thr Tyr Pro Val 180 185 190 Arg Gln Asn Gly Lys Cys Met Ala Lys Met Ile Leu Ser Val Trp Leu 195 200 205 Leu Ser Ala Ser Ile Thr Leu Pro Pro Leu Phe Gly Trp Ala Gln Asn 210 215 220 Val Asn Asp Asp Lys Val Cys Leu Ile Ser Gln Asp Phe Gly Tyr Thr 225 230 235 240 Ile Tyr Ser Thr Ala Val Ala Phe Tyr Ile Pro Met Ser Val Met Leu 245 250 255 Phe Met Tyr Tyr Gln Ile Tyr Lys Ala Ala Arg Lys Ser Ala Ala Lys 260 265 270 His Lys Phe Pro Gly Phe Pro Arg Val Glu Pro Asp Ser Val Ile Ala 275 280 285 Leu Asn Gly Ile Val Lys Leu Gln Lys Glu Val Glu Glu Cys Ala Asn 290 295 300 Leu Ser Arg Leu Leu Lys His Glu Arg Lys Asn Ile Ser Ile Phe Lys 305 310 315 320 Arg Glu Gln Lys Ala Ala Thr Thr Leu Gly Ile Ile Val Gly Ala Phe 325 330 335 Thr Val Cys Trp Leu Pro Phe Phe Leu Leu Ser Thr Ala Arg Pro Phe 340 345 350 Ile Cys Gly Thr Ser Cys Ser Cys Ile Pro Leu Trp Val Glu Arg Thr 355 360 365 Phe Leu Trp Leu Gly Tyr Ala Asn Ser Leu Ile Asn Pro Phe Ile Tyr 370 375 380 Ala Phe Phe Asn Arg Asp Leu Arg Thr Thr Tyr Arg Ser Leu Leu Gln 385 390 395 400 Cys Gln Tyr Arg Asn Ile Asn Arg Lys Leu Ser Ala Ala Gly Met His 405 410 415 Glu Ala Leu Lys Leu Ala Glu Arg Pro Glu Arg Pro Glu Phe Val Leu 420 425 430 Gln Asn Ala Asp Tyr Cys Arg Lys Lys Gly His Asp Ser 435 440 445 3 24 DNA Artificial Sequence misc_feature ()..() primer 3 tgaccctgtg cgtgatcagc attg 24 4 30 DNA Artificial Sequence misc_feature ()..() primer 4 gctttctgtt ctcgcttaaa gatggagatg 30 5 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 5 cctgaatggc atagtgaagc tccagaagga ggtggaagag tgtgc 45 6 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 6 atgcttgagg agtctcgaaa ggtttgcaca ctcttccacc tcctt 45 7 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 7 agcattgaca ggtaccttgg gatcacaagg cccctcacat accct 45 8 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 8 ccatgcattt cccattctgc ctcacagggt atgtgagggg ccttg 45 9 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 9 gtcagcgtca ccgacctcat cgggggcaag tggatctttg gacac 45 10 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 10 tggcgatgaa gacattacag aaaaagtgtc caaagatcca cttgc 45 11 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 11 ggcgccgacc cggtcgcggg ctcctgggca ccgcacctgc tgagc 45 12 45 DNA Artificial Sequence misc_feature ()..() oligonucleotide probe 12 tgggcgccgg gctggctgtc acctcgctca gcaggtgcgg tgccc 45 13 422 PRT Homo Sapiens 13 Met Asp Val Leu Ser Pro Gly Gln Gly Asn Asn Thr Thr Ser Pro Pro 1 5 10 15 Ala Pro Phe Glu Thr Gly Gly Asn Thr Thr Gly Ile Ser Asp Val Thr 20 25 30 Val Ser Tyr Gln Val Ile Thr Ser Leu Leu Leu Gly Thr Leu Ile Phe 35 40 45 Cys Ala Val Leu Gly Asn Ala Cys Val Val Ala Ala Ile Ala Leu Glu 50 55 60 Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Ile Gly Ser Leu Ala Val 65 70 75 80 Thr Asp Leu Met Val Ser Val Leu Val Leu Pro Met Ala Ala Leu Tyr 85 90 95 Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Val Thr Cys Asp Leu Phe 100 105 110 Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Ser Ile Leu His Leu Cys 115 120 125 Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asp Pro Ile Asp Tyr 130 135 140 Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Ala Leu Ile Ser Leu Thr 145 150 155 160 Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro Pro Met Leu Gly Trp Arg 165 170 175 Thr Pro Glu Asp Arg Ser Asp Pro Asp Ala Cys Thr Ile Ser Lys Asp 180 185 190 His Gly Tyr Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile Pro Leu 195 200 205 Leu Leu Met Leu Val Leu Tyr Gly Arg Ile Phe Arg Ala Ala Arg Phe 210 215 220 Arg Ile Arg Lys Thr Val Lys Lys Val Glu Lys Thr Gly Ala Asp Thr 225 230 235 240 Arg His Gly Ala Ser Pro Ala Pro Gln Pro Lys Lys Ser Val Asn Gly 245 250 255 Glu Ser Gly Ser Arg Asn Trp Arg Leu Gly Val Glu Ser Lys Ala Gly 260 265 270 Gly Ala Leu Cys Ala Asn Gly Ala Val Arg Gln Gly Asp Asp Gly Ala 275 280 285 Ala Leu Glu Val Ile Glu Val His Arg Val Gly Asn Ser Lys Glu His 290 295 300 Leu Pro Leu Pro Ser Glu Ala Gly Pro Thr Pro Cys Ala Pro Ala Ser 305 310 315 320 Phe Glu Arg Lys Asn Glu Arg Asn Ala Glu Ala Lys Arg Lys Met Ala 325 330 335 Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu Gly Ile Ile Met Gly 340 345 350 Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Ile Val Ala Leu Val Leu 355 360 365 Pro Phe Cys Glu Ser Ser Cys His Met Pro Thr Leu Leu Gly Ala Ile 370 375 380 Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Leu Asn Pro Val Ile Tyr 385 390 395 400 Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala Phe Lys Lys Ile Ile Lys 405 410 415 Cys Leu Phe Cys Arg Gln 420 14 377 PRT Homo Sapiens 14 Met Ser Pro Leu Asn Gln Ser Ala Glu Gly Leu Pro Gln Glu Ala Ser 1 5 10 15 Asn Arg Ser Leu Asn Ala Thr Glu Thr Ser Glu Ala Trp Asp Pro Arg 20 25 30 Thr Leu Gln Ala Leu Lys Ile Ser Leu Ala Val Val Leu Ser Val Ile 35 40 45 Thr Leu Ala Thr Val Leu Ser Asn Ala Phe Val Leu Thr Thr Ile Leu 50 55 60 Leu Thr Arg Lys Leu His Thr Pro Ala Asn Tyr Leu Ile Gly Ser Leu 65 70 75 80 Ala Thr Thr Asp Leu Leu Val Ser Ile Leu Val Met Pro Ile Ser Ile 85 90 95 Ala Tyr Thr Ile Thr His Thr Trp Asn Phe Gly Gln Ile Leu Cys Asp 100 105 110 Ile Trp Leu Ser Ser Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu His 115 120 125 Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asp Ala Leu 130 135 140 Glu Tyr Ser Lys Arg Arg Thr Ala Gly His Ala Ala Thr Met Ile Ala 145 150 155 160 Ile Val Trp Ala Ile Ser Ile Cys Ile Ser Ile Pro Pro Leu Phe Trp 165 170 175 Arg Gln Ala Lys Ala Gln Glu Glu Met Ser Asp Cys Leu Val Asn Thr 180 185 190 Ser Gln Ile Ser Tyr Thr Ile Tyr Ser Thr Cys Gly Ala Phe Tyr Ile 195 200 205 Pro Ser Val Leu Leu Ile Ile Leu Tyr Gly Arg Ile Tyr Arg Ala Ala 210 215 220 Arg Asn Arg Ile Leu Asn Pro Pro Ser Leu Tyr Gly Lys Arg Phe Thr 225 230 235 240 Thr Ala His Leu Ile Thr Gly Ser Ala Gly Ser Ser Leu Cys Ser Leu 245 250 255 Asn Ser Ser Leu His Glu Gly His Ser His Ser Ala Gly Ser Pro Leu 260 265 270 Phe Phe Asn His Val Lys Ile Lys Leu Ala Asp Ser Ala Leu Glu Arg 275 280 285 Lys Arg Ile Ser Ala Ala Arg Glu Arg Lys Ala Thr Lys Ile Leu Gly 290 295 300 Ile Ile Leu Gly Ala Phe Ile Ile Cys Trp Leu Pro Phe Phe Val Val 305 310 315 320 Ser Leu Val Leu Pro Ile Cys Arg Asp Ser Cys Trp Ile His Pro Ala 325 330 335 Leu Phe Asp Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn 340 345 350 Pro Ile Ile Tyr Thr Val Phe Asn Glu Glu Phe Arg Gln Ala Phe Gln 355 360 365 Lys Ile Val Pro Phe Arg Lys Ala Ser 370 375 15 390 PRT Homo Sapiens 15 Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pro Pro Pro Ala Gly Ser 1 5 10 15 Glu Thr Trp Val Pro Gln Ala Asn Leu Ser Ser Ala Pro Ser Gln Asn 20 25 30 Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp Ser Ile Ser Leu Pro Trp 35 40 45 Lys Val Leu Leu Val Met Leu Leu Ala Leu Ile Thr Leu Ala Thr Thr 50 55 60 Leu Ser Asn Ala Phe Val Ile Ala Thr Val Tyr Arg Thr Arg Lys Leu 65 70 75 80 His Thr Pro Ala Asn Tyr Leu Ile Ala Ser Leu Ala Val Thr Asp Leu 85 90 95 Leu Val Ser Ile Leu Val Met Pro Ile Ser Thr Met Tyr Thr Val Thr 100 105 110 Gly Arg Trp Thr Leu Gly Gln Val Val Cys Asp Phe Trp Leu Ser Ser 115 120 125 Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu His Leu Cys Val Ile Ala 130 135 140 Leu Asp Arg Tyr Trp Ala Ile Thr Asp Ala Val Glu Tyr Ser Ala Lys 145 150 155 160 Arg Thr Pro Lys Arg Ala Ala Val Met Ile Ala Leu Val Trp Val Phe 165 170 175 Ser Ile Ser Ile Ser Leu Pro Pro Phe Phe Trp Arg Gln Ala Lys Ala 180 185 190 Glu Glu Glu Val Ser Glu Cys Val Val Asn Thr Asp His Ile Leu Tyr 195 200 205 Thr Val Tyr Ser Thr Val Gly Ala Phe Tyr Phe Pro Thr Leu Leu Leu 210 215 220 Ile Ala Leu Tyr Gly Arg Ile Tyr Val Glu Ala Arg Ser Arg Ile Leu 225 230 235 240 Lys Gln Thr Pro Asn Arg Thr Gly Lys Arg Leu Thr Arg Ala Gln Leu 245 250 255 Ile Thr Asp Ser Pro Gly Ser Thr Ser Ser Val Thr Ser Ile Asn Ser 260 265 270 Arg Val Pro Asp Val Pro Ser Glu Ser Gly Ser Pro Val Tyr Val Asn 275 280 285 Gln Val Lys Val Arg Val Ser Asp Ala Leu Leu Glu Lys Lys Lys Leu 290 295 300 Met Ala Ala Arg Glu Arg Lys Ala Thr Lys Thr Leu Gly Ile Ile Leu 305 310 315 320 Gly Ala Phe Ile Val Cys Trp Leu Pro Phe Phe Ile Ile Ser Leu Val 325 330 335 Met Pro Ile Cys Lys Asp Ala Cys Trp Phe His Leu Ala Ile Phe Asp 340 345 350 Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Ile Ile 355 360 365 Tyr Thr Met Ser Asn Glu Asp Phe Lys Gln Ala Phe His Lys Leu Ile 370 375 380 Arg Phe Lys Cys Thr Ser 385 390 16 365 PRT Homo Sapiens 16 Met Asn Ile Thr Asn Cys Thr Thr Glu Ala Ser Met Ala Ile Arg Pro 1 5 10 15 Lys Thr Ile Thr Glu Lys Met Leu Ile Cys Met Thr Leu Val Val Ile 20 25 30 Thr Thr Leu Thr Thr Leu Leu Asn Leu Ala Val Ile Met Ala Ile Gly 35 40 45 Thr Thr Lys Lys Leu His Gln Pro Ala Asn Tyr Leu Ile Cys Ser Leu 50 55 60 Ala Val Thr Asp Leu Leu Val Ala Val Leu Val Met Pro Leu Ser Ile 65 70 75 80 Ile Tyr Ile Val Met Asp Arg Trp Lys Leu Gly Tyr Phe Leu Cys Glu 85 90 95 Val Trp Leu Ser Val Asp Met Thr Cys Cys Thr Cys Ser Ile Leu His 100 105 110 Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asn Ala Ile 115 120 125 Glu Tyr Ala Arg Lys Arg Thr Ala Lys Arg Ala Ala Leu Met Ile Leu 130 135 140 Thr Val Trp Thr Ile Ser Ile Phe Ile Ser Met Pro Pro Leu Phe Trp 145 150 155 160 Arg Ser His Arg Arg Leu Ser Pro Pro Pro Ser Gln Cys Thr Ile Gln 165 170 175 His Asp His Val Ile Tyr Thr Ile Tyr Ser Thr Leu Gly Ala Phe Tyr 180 185 190 Ile Pro Leu Thr Leu Ile Leu Ile Leu Tyr Tyr Arg Ile Tyr His Ala 195 200 205 Ala Lys Ser Leu Tyr Gln Lys Arg Gly Ser Ser Arg His Leu Ser Asn 210 215 220 Arg Ser Thr Asp Ser Gln Asn Ser Phe Ala Ser Cys Lys Leu Thr Gln 225 230 235 240 Thr Phe Cys Val Ser Asp Phe Ser Thr Ser Asp Pro Thr Thr Glu Phe 245 250 255 Glu Lys Phe His Ala Ser Ile Arg Ile Pro Pro Phe Asp Asn Asp Leu 260 265 270 Asp His Pro Gly Glu Arg Gln Gln Ile Ser Ser Thr Arg Glu Arg Lys 275 280 285 Ala Ala Arg Ile Leu Gly Leu Ile Leu Gly Ala Phe Ile Leu Ser Trp 290 295 300 Leu Pro Phe Phe Ile Lys Glu Leu Ile Val Gly Leu Ser Ile Tyr Thr 305 310 315 320 Val Ser Ser Glu Val Ala Asp Phe Leu Thr Trp Leu Gly Tyr Val Asn 325 330 335 Ser Leu Ile Asn Pro Leu Leu Tyr Thr Ser Phe Asn Glu Asp Phe Lys 340 345 350 Leu Ala Phe Lys Lys Leu Ile Arg Cys Arg Glu His Thr 355 360 365 17 347 PRT Homo Sapiens 17 Met Pro Ser Lys Ile Leu Val Ser Leu Thr Leu Ser Gly Leu Ala Leu 1 5 10 15 Met Thr Thr Thr Ile Asn Ser Leu Val Ile Ala Ala Ile Ile Val Thr 20 25 30 Arg Lys Leu His His Pro Ala Asn Tyr Leu Ile Cys Ser Leu Ala Val 35 40 45 Thr Asp Phe Leu Val Ala Val Leu Val Met Pro Phe Ser Ile Val Tyr 50 55 60 Ile Val Arg Glu Ser Trp Ile Met Gly Gln Val Val Cys Asp Ile Trp 65 70 75 80 Leu Ser Val Asp Ile Thr Cys Cys Thr Cys Ser Ile Leu His Leu Ser 85 90 95 Ala Ile Ala Leu Asp Arg Tyr Arg Ala Ile Thr Asp Ala Val Glu Tyr 100 105 110 Ala Arg Lys Arg Thr Pro Lys His Ala Gly Ile Met Ile Thr Ile Val 115 120 125 Trp Ile Ile Ser Val Phe Ile Ser Met Pro Pro Leu Phe Trp Arg His 130 135 140 Gln Gly Thr Ser Arg Asp Asp Glu Cys Ile Ile Lys His Asp His Ile 145 150 155 160 Val Ser Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile Pro Leu Ala 165 170 175 Leu Ile Leu Ile Leu Tyr Tyr Lys Ile Tyr Arg Ala Ala Lys Thr Leu 180 185 190 Tyr His Lys Arg Gln Ala Ser Arg Ile Ala Lys Glu Glu Val Asn Gly 195 200 205 Gln Val Leu Leu Glu Ser Gly Glu Lys Ser Thr Lys Ser Val Ser Thr 210 215 220 Ser Tyr Val Leu Glu Lys Ser Leu Ser Asp Pro Ser Thr Asp Phe Asp 225 230 235 240 Lys Ile His Ser Thr Val Arg Ser Leu Arg Ser Glu Phe Lys His Glu 245 250 255 Lys Ser Trp Arg Arg Gln Lys Ile Ser Gly Thr Arg Glu Arg Lys Ala 260 265 270 Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala Phe Val Ile Cys Trp Leu 275 280 285 Pro Phe Phe Val Lys Glu Leu Val Val Asn Val Cys Asp Lys Cys Lys 290 295 300 Ile Ser Glu Glu Met Ser Asn Phe Leu Ala Trp Leu Gly Tyr Leu Asn 305 310 315 320 Ser Leu Ile Asn Pro Leu Ile Tyr Thr Ile Phe Asn Glu Asp Phe Lys 325 330 335 Lys Ala Phe Gln Lys Leu Val Arg Cys Arg Cys 340 345 

What is claimed is:
 1. An isolated nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor.
 2. A nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a human 5-HT_(4B) receptor.
 3. A nucleic acid molecule of claim 1, wherein the nucleic acid molecule is a DNA molecule.
 4. A DNA molecule of claim 3, wherein the DNA molecule is a cDNA molecule.
 5. A DNA molecule of claim 3 wherein the DNA molecule is derived from genomic DNA.
 6. A isolated nucleic acid molecule which has a nucleic acid sequence which differs from the sequence of a nucleic acid molecule encoding a 5-HT_(4B) receptor at one or more nucleotides and which does not encode a protein having 5HT_(4B) receptor activity.
 7. A nucleic acid molecule of claim 6, wherein the nucleic acid molecule is a DNA molecule.
 8. A DNA molecule of claim 7, wherein the DNA molecule is a cDNA molecule.
 9. A vector comprising a cDNA molecule of claim
 4. 10. A plasmid vector of claim
 9. 11. A vector of claim 9 adapted for expression in a bacterial cell which comprises the regulatory elements necessary for expression of the cDNA encoding a 5-HT_(4B) receptor in the bacterial cell so located relative to the cDNA as to permit expression thereof.
 12. A vector of claim 9 adapted for expression in a yeast cell which comprises the regulatory elements necessary for the expression of the cDNA encoding a 5-HT_(4B) receptor in the yeast cell so located relative to the cDNA as to permit expression thereof.
 13. A vector of claim 9 adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the cDNA encoding a 5-HT_(4B) receptor in the mammalian cell so located relative to the cDNA as to permit expression thereof.
 14. A plasmid of claim 10 adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the DNA in the mammalian cell so located relative to the DNA encoding a 5-HT_(4B) receptor as to permit expression thereof.
 15. A plasmid of claim 14 designated pcEXV-5HT_(4B) (ATCC. Accession No. 75332).
 16. A mammalian cell comprising the plasmid of claim
 10. 17. A mammalian cell of claim 16, wherein the mammalian cell is an LM (tk−) cell.
 18. An LM (tk−) cell comprising the plasmid of claim 14 designated L-5-HT_(4B) (ATCC Accession No. 11166).
 19. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a mammalian 5-HT_(4B) receptor.
 20. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT_(4B) receptor.
 21. The nucleic acid probe of claim 19, wherein the nucleic acid is DNA.
 22. A mixture of nucleic acid probes in accordance with claim 19, such probes having sequences which differ from one another at predefined positions.
 23. An antisense oligonucleotide having a sequence capable of specifically binding to a mRNA molecule encoding a mammalian 5-HT_(4B) receptor so as to prevent translation of the mRNA molecule.
 24. An antisense oligonucleotide capable of specifically binding to a mRNA molecule encoding a human 5-HT_(4B) receptor so as to prevent translation of the mRNA molecule.
 25. An antisense oligonucleotide of claim 23 comprising chemical analogs of nucleotides.
 26. A mixture of antisense oligonucleotides according to claim 23, such oligonucleotides having sequences which differ from one another at predefined positions.
 27. A method for detecting expression of a mammalian 5-HT_(4B) receptor, which comprises obtaining RNA from cells or tissue, contacting the RNA so obtained with a nucleic acid probe of claim 19 under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of the mammalian 5-HT_(4B) receptor, and thereby detecting the expression of the mammalian 5-HT_(4B) receptor.
 28. A method for detecting expression of a human 5-HT_(4B) receptor, which comprises obtaining RNA from cells or tissue, contacting the RNA so obtained with a nucleic acid probe of claim 20 under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of the human 5-HT_(4B) receptor, and thereby detecting the expression of the human 5-HT_(4B) receptor.
 29. A method of detecting expression of a mammalian 5-HT_(4B) receptor in a cell or tissue by in situ hybridization, which comprises contacting the cell or tissue with a nucleic acid probe of claim 19 under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of a mammalian 5-HT_(4B) receptor, and thereby detecting the expression of a mammalian 5-HT_(4B) receptor.
 30. A method of detecting expression of a human 5-HT_(4B) receptor in a cell or tissue by in situ hybridization, which comprises contacting the cell or tissue with a nucleic acid probe of claim 20 under hybridizing conditions, detecting the presence of any mRNA hybridized to the probe, the presence of mRNA hybridized to the probe indicating expression of a human 5-HT_(4B) receptor, and thereby detecting the expression of the human 5-HT_(4B) receptor.
 31. A method of isolating from a gene library a gene encoding a receptor other than the 5HT_(4B) receptor which comprises contacting the library under hybridizing conditions with a probe of claim 21 and isolating any gene to which the probe hybridizes.
 32. A method of claim 31, which additionally comprises simultaneously contacting the DNA comprising the library under hybridizing conditions with a second nucleic acid probe comprising a sequence capable of hybridizing to a DNA sequence of the complementary strand of the DNA of the gene to which the first probe hybridizes, treating any gene sequence to which both probes hybridized so as to produce multiple copies of the gene sequence, isolating the amplified gene sequence and using the isolated gene sequence as a probe to isolate from a gene library the gene to which the amplified DNA sequence hybridizes.
 33. The gene isolated by the method of claim 31 or
 32. 34. A synthetic gene which comprises the isolated nucleic molecule of claim 1 and at least one regulatory element attached thereto so as to increase the number of RNA molecules transcribed from the synthetic gene.
 35. A synthetic gene which comprises the isolated nucleic acid molecule of claim 1 and at least one regulatory element attached thereto so as to decrease the number of RNA molecules transcribed from the synthetic gene.
 36. An isolated mammalian 5-HT_(4B) receptor protein.
 37. The receptor protein of claim 36, wherein the mammalian 5-HT_(4B) receptor protein is a human 5-HT_(4B) receptor.
 38. A method of preparing a mammalian 5-HT_(4B) receptor of claim 36, which comprises inducing cells to express the mammalian 5-HT_(4B) receptor and recovering the mammalian 5-HT_(4B) receptor from the resulting cells.
 39. A method of preparing a mammalian 5-HT_(4B) receptor protein of claim 36, which comprises inserting a nucleic acid molecule encoding the mammalian 5-HT_(4B) receptor in a suitable vector, inserting the resulting vector in suitable host cell and recovering the mammalian 5-HT_(4B) receptor produced by the resulting cell.
 40. A method of preparing a human 5-HT_(4B) receptor of claim 37, which comprises inducing cells to express the human 5-HT_(4B) receptor and recovering the human 5-HT_(4B) receptor from the resulting cells.
 41. A method of preparing a human 5-HT_(4B) receptor protein of claim 37, which comprises inserting a nucleic acid molecule encoding the human 5-HT_(4B) receptor in a suitable vector, inserting the resulting vector in suitable host cell and recovering the human 5-HT_(4B) receptor produced by the resulting cell.
 42. An antibody directed to a mammalian 5-HT_(4B) receptor or to a protein fragment of the mammalian 5-HT_(4B) receptor.
 43. An antibody directed to a human 5-HT_(4B) receptor or to a protein fragment of the human 5-HT_(4B) receptor.
 44. An antibody of claim 42, wherein the antibody is a monoclonal antibody.
 45. An antibody of claim 43, wherein the antibody is a monoclonal antibody.
 46. A monoclonal antibody of claim 44, wherein the antibody is directed to an epitope of a mammalian cell-surface 5-HT_(4B) receptor and having an amino acid sequence substantially the same as the amino acid sequence of a cell-surface epitope of the mammalian 5-HT_(4B) receptor.
 47. A monoclonal antibody of claim 45, wherein the antibody is directed to an epitope of a human cell-surface 5-HT_(4B) receptor and having an amino acid sequence substantially the same as the amino acid sequence for a cell-surface epitope of the human 5-HT_(4B) receptor.
 48. A pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.
 49. A pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.
 50. A pharmaceutical composition comprising an effective amount of an oligonucleotide of claim 24 effective to reduce expression of a human 5-HT_(4B) receptor by passing through a cell membrane and specifically binding with mRNA encoding a human 5-HT_(4B) receptor in the cell so as to prevent its translation and a pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane.
 51. A pharmaceutical composition claim 50, wherein the nucleotide is coupled to a substance which inactivates mRNA.
 52. A pharmaceutical composition of claim 51, wherein the substance which inactivates the mRNA is a ribozyme.
 53. A pharmaceutical composition of claim 51, wherein the pharmaceutically acceptable hydrophobic carrier capable of passing through a cell membrane comprises a structure which binds to a transporter specific for a selected cell type and is thereby taken up by the cells of the selected cell type.
 54. A pharmaceutical composition which comprises an amount of the antibody of claim 43 effective to block binding of naturally occurring substrates to a human 5-HT_(4B) receptor and a pharmaceutically acceptable carrier.
 55. A transgenic nonhuman mammal which comprises a nucleic acid molecule of claim
 1. 56. A transgenic nonhuman mammal which comprises the nucleic acid molecule of claim
 6. 57. A transgenic nonhuman mammal whose genome comprises a nucleic acid molecule of claim 1 so placed as to be transcribed into antisense mRNA complementary to mRNA encoding a human 5-HT_(4B) receptor and which hybridizes to mRNA encoding a human 5-HT_(4B) receptor thereby reducing its translation.
 58. The transgenic nonhuman mammal of claim 55, wherein the nucleic acid molecule further comprises an inducible promoter.
 59. The transgenic nonhuman mammal of claim 55 or 58 wherein the nucleic molecule additionally comprises tissue specific regulatory elements.
 60. The transgenic non-human mammal of 55 wherein the transgenic non-human mammal is a mouse.
 61. A method for determining the physiological effects of varying the levels of expression of a human 5-HT_(4B) receptor which comprises producing a transgenic non-human mammal whose levels of expression of a human 5-HT_(4B) receptor can be varied by use of an inducible promoter.
 62. A method for determining the physiological effects of expressing varying levels of a human 5-HT_(4B) receptor which comprises producing a panel of transgenic non-human mammals each expressing a different amount of a human 5-HT_(4B) receptor.
 63. A method for determining whether a compound not known to be capable of specifically binding to a human 5-HT_(4B) receptor can specifically bind to the human 5-HT_(4B) receptor, which comprises contacting a mammalian cell comprising a plasmid adapted for expression in a mammalian cell which plasmid further comprises DNA which expresses a human 5-HT_(4B) receptor on the cell's surface with the compound under conditions permitting binding of ligands known to bind to a human 5-HT_(4B) receptor, detecting the presence of any compound bound to the human 5-HT_(4B) receptor, the presence of bound compound indicating that the compound is capable of specifically binding to the human 5-HT_(4B) receptor.
 64. The method of claim 63, wherein the mammalian cell is a non-neuronal cell.
 65. A method of screening compound to identify drugs which interact with, and specifically bind to, a human 5-HT_(4B) receptor on the surface of a cell, which comprises contacting a mammalian cell which comprises a plasmid adapted for expression in a mammalian cell which plasmid further comprises DNA which expresses a human 5-HT_(4B) receptor on the cell's surface with a plurality of compounds, determining those compounds which bind to the human 5-HT_(4B) receptor expressed on the cell surface of the mammalian cell, and thereby identifying compounds which interact with, and specifically bind to, the human 5-HT_(4B) receptor.
 66. The method of claim 65, wherein the mammalian cell is a non-neuronal cell.
 67. A method for determining whether a compound not known to be capable of specifically binding to a human 5-HT_(4B) receptor can specifically bind to a human 5-HT_(4B) receptor, which comprises preparing a cell extract from mammalian cells, which comprise a plasmid adapted for expression in a mammal, which plasmid further comprises DNA which expresses a human 5-HT_(4B) receptor on the cell's surface, isolating a membrane fraction from the cell extract, incubating the compound with the membrane fraction under conditions permitting binding of ligands known to bind to the human 5-HT_(4B) receptor, detecting the presence of any bound compound, and thereby determining whether the compound is capable of specifically binding to the human 5-HT_(4B) receptor.
 68. The method of claim 67, wherein the mammalian cell is a non-neuronal cell.
 69. A method for screening compounds to identify drugs that interact with, and specifically bind to, a human 5-HT_(4B) receptor, which comprises preparing a cell extract from mammalian cells, which comprise a plasmid adapted for expression in a mammalian cell which plasmid further comprises DNA which expresses a human 5-HT_(4B) receptor on the cell's surface, isolating a membrane fraction from the cell extract, incubating the membrane fraction with a plurality of compounds, determining those compounds which interact with and bind to the human 5-HT_(4B) receptor, and thereby identifying compounds which interact with, and specifically bind to, the human 5-HT_(4B) receptor.
 70. The method of claim 69, wherein the mammalian cell is a non-neuronal cell.
 71. A method for identify a compound which is not known to be capable of binding to the human 5-HT_(4B) receptor activates the human 5-HT_(4B) receptor on the surface of a mammalian cell or prevents a ligand which does so, which comprises contacting the mammalian cell which cell comprises a plasmid adapted for expression in the mammalian cell such plasmid further comprising DNA which expresses the human 5-HT_(4B) receptor on the cell surface of the mammalian cell with the compound under conditions permitting activation of blockade of a functional response, determining whether the compound activates the human 5-HT_(4B) receptor or prevents a ligand which does so, and thereby identifying the compound as a compound which interacts with, and activates the human 5-HT_(4B) receptor or prevents the activation of the human 5-HT_(4B) receptor by a ligand which does so.
 72. The method of claim 71, wherein the mammalian cell is a non-neuronal cell comprising the cellular components necessary to produce a second messenger and wherein the determination of whether the compound activates of blocks the activation of the human 5-HT_(4B) comprises detecting the change in the concentration of the second messenger.
 73. The method of claim 72, wherein the second messenger is cyclic AMP (cAMP).
 74. The method of claim 72, wherein the non-neuronal cell is a COS-7 cell.
 75. A method of claim 72, wherein the second messenger is an inositol phosphate metabolite.
 76. The method of claim 72, wherein the second messenger is intracellular calcium.
 77. A compound identified by the methods of claims 63, 67, 71, 72, 73, 75 or
 76. 78. A pharmaceutical composition of a drug identified by the methods of claims 65, 69, 71, 72, 73, 75, or
 76. 79. A method for detecting the presence of a human 5-HT_(4B) receptor on the surface of a cell, which comprises contacting the cell with an antibody of claim 44, under conditions that permit binding of the antibody to the receptor, detecting the presence of any of the antibody bound to the cell, and thereby the presence of a human 5-HT_(4B) receptor on the surface of the cell.
 80. A method for treating an abnormal condition related to an excess of activity of a human 5-HT_(4B) receptor, which comprises administering a patient an amount of a pharmaceutical composition of claim 82, effective to reduce 5-HT_(4B) activity as a result of naturally occurring substrate binding to and activating the 5-HT_(4B) receptor.
 81. A method for diagnosing a predisposition to a disorder associated with the expression of a human 5-HT_(4B) receptor allele which comprises: a. obtaining DNA from subjects suffering from a disorder; b. performing a restriction digest of the DNA with a panel of restriction enzymes; c. electrophoretically separating the resulting DNA fragments on a sizing gel; d. contacting the gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a human 5-HT_(4B) receptor and labelled with a detectable marker; e. detecting the labelled bands which have hybridized to the DNA encoding 5-HT_(4B) receptor, labelled with the detectable marker to create a unique band pattern specific to the DNA of subjects suffering with the disorder; f. preparing DNA for diagnosis by steps a-e; and g. comparing the unique band pattern specific to the DNA of patients suffering from the disorder from step e and DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
 82. The method of treating abnormalities which are alleviated by an increase in the activity of a 5-HT_(4B) receptor, which comprises administering a patient an amount of a pharmaceutical composition of claim 81, effective to increase the activity of the 5-HT_(4B) receptor thereby alleviating abnormalities resulting from abnormally low receptor activity.
 83. The method of claim 81, wherein a disorder is associated with the expression of a specific human 5-HT_(4B) receptor allele is diagnosed.
 84. A method of identifying a substance capable of alleviating the abnormalities resulting from overexpression of a human 5-HT_(4B) receptor which comprises administering a substance to the transgenic non-human mammal of claim 61 or 62, and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overexpression of the human 5-HT_(4B) receptor.
 85. A method of identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human 5-HT_(4B) receptor, which comprises administering a substance to the transgenic mammal of claim 56 or 57, and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underexpression of the human 5-HT_(4B) receptor.
 86. A method of treating abnormalities in a subject, wherein the abnormality is alleviated by the reduced expression of a human 5-HT_(4B) receptor which comprises administering to a subject an effective amount of the pharmaceutical composition of claim 48, 50, 77 or 78 effective to reduce expression of the human 5-HT_(4B) receptor.
 87. A method of treating abnormalities resulting from underexpression of a human 5-HT_(4B) receptor which comprises administering to a subject an amount of a pharmaceutical composition of claim 49, 77 or 78 effective to alleviate abnormalities resulting from underexpression of the human 5-HT_(4B) receptor.
 88. A vector of claim 9 adapted for expression in an insect cell which comprises the regulatory elements necessary for expression of the cDNA encoding a 5-HT_(4B) receptor in the insect cell so located relative to the cDNA as to permit expression thereof. 